Method for screening for the presence of genetic defect associated with thrombosis and/or poor anticoagulant response to activated protein C

ABSTRACT

Method for screening for the presence of a genetic defect associated with thrombosis and/or poor anticoagulant response to activated protein C (APC). The method is directed at detecting one or more mutations at one or more of the cleavage and/or binding sites for APC of Factor V and/or Factor Va or at Factor VIII and/or Factor VIIIa at either nucleic acid or protein level or both.

RELATED APPLICATIONS

The present application is a continued prosecution application of U.S.application Ser. No. 09/165,019 filed Sep. 30, 1998, which is adivisional of commonly assigned U.S. patent application Ser. No.08/454,353 entitled A Method for Screening for the Presence of GeneticDefect Associated with Thrombosis and/or Poor Anticoagulant Response toActivated Protein C filed Jun. 6, 1995, the disclosure of which isincorporated by reference as if set forth fully, which claims priorityfrom European Patent Application Serial No. 94200377.3 filed Feb. 14,1994.

The subject invention lies in the field of haemostasis and in particularis directed at the aspect of thrombosis. More particularly the inventionis directed at a method for screening and diagnosis of thrombophilia,especially hereditary thrombophilia. The method according to theinvention can then be used for determining the risk for thrombosis inindividuals.

BACKGROUND TO THE INVENTION

Deep vein thrombosis is a common disease. Well established risk factorsinclude recent surgery, malignant disorders, pregnancy and labour, longterm immobilisation, and deficiency of one of the main inhibitors of theclotting system (Ref. 1). The main inhibitors are known to be protein C,protein S and antithrombin. The causes of deep vein thrombosis in manypatients remain unclear. It has recently been established however that apoor anticoagulant response to activated protein C (APC) is present inseveral families with a hereditary tendency to venous thrombosis (Ref.2).

The anticoagulant property of APC resides in its capacity to inactivatethe activated cofactors Va and VIIIa by limited proteolysis (Ref. 3).This inactivation of cofactors Va and VIIIa results in reduction of therate of formation of thrombin, the key enzyme of coagulation. In vitro,this effect can be visualised by adding APC to normal plasma andaccordingly determining the effect thereof in a coagulation test, forexample in a test determining the APTT (activated partial thromboplastintime). Activation of protein C occurs at the surface of endothelialcells via the thrombin-thrombomodulin complex (Ref. 27). Thrombomodulinis a membrane glycoprotein that can bind thrombin. By this bindingthrombin loses the ability to convert fibrinogen to fibrin and theability to activate blood platelets. In other words thrombin loses itscoagulant properties and reduces its further own production (so-callednegative feed-back) by activating protein C. In vivo (in the presence ofcalcium) the activation of protein C is almost completely dependent onthe availability of thrombomodulin on the endothelium. APC issubsequently neutralized by formation of complexes with APC inhibitor(PCI) and α₁ antitrypsin, which means that in normal conditions itremains only for a short time in the circulation and the anticoagulanteffect remains generally locally expressed.

It was generally accepted that the inactivation of the cofactors Va andVIIIa by APC proceeds only optimally in the presence of Ca²⁺,phospholipids and the APC cofactor protein S (Ref. 4, 28, 29). Morerecently this view was, however, challenged by the finding that insystems of purified proteins protein S has little cofactor activity toAPC (Ref. 5, Ref. 6). A possible solution for this apparent discrepancybetween the observations in vivo (thrombotic tendency in hereditaryprotein S deficiency) and in vitro (poor APC cofactor activity ofprotein S in systems of purified proteins) could be offered by thefinding of Dahlbäck et al (Ref. 2) who reported patients with normalvalues for antithrombin activity, protein C (immunologically andfunctionally) and protein S (immunologically and functionally) withoutindications for abnormal plasminogen, abnormal fibrinogen or lupusanticoagulants, but with a reduced anticoagulant response to activatedprotein C. The latter was found with a new test developed by Dahlback(Ref. 2) in which he studies the response (coagulation time, APTT) of aplasma after in vitro addition of purified human APC. The addition ofactivated protein C to the plasma of these thrombotic patients did notresult in the expected prolongation of the activated partialthromboplastin time (APTT). After postulating a number of mechanisms forthis phenomenon only one was considered to provoke the pooranticoagulant response to APC, namely the existence of a hithertounknown cofactor to APC that is deficient in these patients.

The following mechanisms have to date been rejected as being causes ofthe poor anticoagulant response to APC:

1. The presence of an auto antibody against APC

2. A fast acting protease inhibitor reacting with APC

3. A functional protein S deficiency

4. Mutations in the Factor V or Factor VIII gene

Dahlbäck (Ref. 2, 7) postulated that in the families studied ahereditary shortage of a hereto unknown APC cofactor that purportedlyworks independently of protein S was the cause of APC resistance.Dahlbäck et al (Ref. 2) also described a test method for diagnosing thethrombo embolic disorders by addition of activated protein C to apatient sample containing coagulation factors followed by measurement ofan enzyme activity that is influenced by the addition of APC in aninternational patent application WO93/10261. It is stated in theapplication of Dahlbäck et al that the experimental results presentedindicated that the disorders in question are related to a hithertounknown coagulation factor or factors or unknown interactions of knownfactors. The unknown factor is not Factor Va or VIIIa that is resistantto degradation by APC and neither is it an inhibitor of theimmunoglobulin type of APC. Furthermore it is not related to protein Sdeficiency. Dahlbäck et al (Ref. 2) state that their invention is amethod particularly useful for further diagnosis of thromboembolicdiseases such as hereditary or non hereditary thrombophilia and fordetermining a risk for thrombosis in connection with pregnancy, takinganticonception pills, surgery etc. They describe their method as beingcharacterized in that the coagulation system in a sample is activated,wholly or partly in a manner known per se and incubated with activatedprotein C, whereupon a substrate conversion reaction rate like clottingor conversion of a chromogenic substrate is determined. The conversionrate obtained is compared with values obtained for normal plasmasamples. If the rate is enhanced it indicates that the individual fromwhich the sample is derived may suffer from a clotting disease. Thedisease is not expressed by protein S deficiency or by formation ofFactor Va or Factor VIIIa resistant to degradation by APC or by aninhibitor of the immunoglobulin type for APC. In the internationalapplication it is also stated by Dahlbäck et al that the data in theapplication indicated that the patient in question could not carry adefective Factor VIII/VIIIa in contrast to what they had previouslystated in Thromb. Haemostas. 65, Abstract 39, 658 (1991), whereinaddition of activated protein C to a plasma sample of a patient, andstudy of the effect produced was claimed to have illustrated a defectiveFactor VIIIa molecule not degraded by activated protein C. Furthermorein the international patent application the assay was used to directlymeasure the inhibition of Factors Va and VIIIa by APC. Using the FactorXa based clotting assay described therein, the inhibition of patientFactor Va by APC was found to be normal suggesting that Factor Va in thepatient's plasma was degraded in a normal fashion by exogeneously addedAPC.

Following the publication by Dahlbäck et al (Ref. 2) other groupsstarted research in this area. In Blood Vol. 82, nr. 7, 1993 on pages1989-1993 Griffin et al describe the results of APC resistance testscarried out among 25 venous thrombotic patients with no identifiableblood coagulation abnormality and 22 patients previously identified withheterozygous protein C or protein S deficiency. The APC inducedprolongation of the activated partial thromboplastin time assay forthese patients was compared with results for 35 normal subjects. Theresults showed that his new defect in antiocoagulant response to APC wassurprisingly present in 52 to 64% of the 25 patients i.e. in themajority of previously undiagnosed thrombophilia cases. The deficiencywas not present in 20 of 22 heterozygous protein C or protein Sdeficient patients. This suggested that the new factor is a risk factorindependent of protein C or protein S deficiency. Mixing of normal bloodplasma with each of two extremely defective plasmas (APC-inducedprolongation of APTT<20 s) was performed and the APTT assays were madeto assess the ability of normal plasma to correct the poor response ofthe defective plasmas. The results were similar to those of Dahlbäck etal (Ref. 2). This also suggested that normal plasma contains a factorwhich is missing from the defective patients plasmas. Values are givenin the article for the net calculated prolongation in APTT, simplydefined as an APTT value in the presence of APC minus the APTT value inthe absence of APC. The article also describes the ratio of the APTTwith APC to the APTT without APC and the fact that this parameter wascompared to values for the APC induced APTT prolongation. Thiscomparison indicated an excellent correlation between these parametersfor the normals, with an extremely low APTT ratio value being indicativeof abnormal patients. Therefore it followed that either the parameter ofAPC-induced APTT prolongation or the parameter of the ratio of APTTvalues with versus without APC or both parameters can be used asdiagnostic parameter. None of these parameters seemed more useful forthis purpose than the other according to the article. Furthermore in thearticle it was stated that the APC-induced prolongation of the APTTassay used was reminiscent of the assay involving APC-inducedinactivation of endogenous Factor VIII in the plasmas of patients withlupus anticoagulant reported by Potzsch et al (Ref. 19) in Blood 80:267a 1992 (Abstract)). Based on this latter assay it was reported in theGriffin et al article that plasma from lupus anticoagulant patients withthrombosis gave a poor response to APC and that patients with thrombosiscould thereby be distinguished from those without thrombosis. Griffin etal speculated that auto-antibodies against the new hypothesizedAPC-cofactor could play a role in the risk of thrombosis among patientswith lupus anticoagulants. It is further stated by them that it istempting to speculate that an acquired deficiency of the newAPC-cofactor could be associated with an acquired risk of thrombosis.

In the Lancet, Dec. 18, 1993, Vol. 342, on pages 1503-1506 Koster et alhave elaborated further on the link between APC-resistance andthrombosis by describing how a population based case control study wasundertaken to test the clinical importance of the abnormality in thecoagulation system that is characterized by a poor antiocoagulantresponse to activated protein C (APC). From studies within families thispoor response to APC appears to inherit as an autosomal dominant trait(Ref. 2, 7 and 47). Among patients referred to a coagulation unitbecause of unexplained thrombosis this abnormality was a major cause ofthrombophilia with a prevalence of about 40% (Ref. 8 and 9). In thestudy described by Koster et al in the Lancet, Dec. 18, 1993, Vol. 342,pages 1503-1506, the clinical importance of this poor response to APCwas investigated in unselected consecutive patients, aged less than 70years, with a first objectively confirmed episode of deep veinthrombosis and without an underlying malignancy. The sensitivity ofthese patients plasma to APC was compared with that of matched healthycontrols. The sensitivity of their plasma APTT to activated protein C(APC) was measured essentially as described by Dahlbäck et al (Ref. 2)using the reagents and reaction conditions previously developed for theprotein S activity assay (Ref. 11). The results were expressed as APCsensitivity ratios (APC-SR) defined as the value of APTT (+APC) over theAPTT (−APC). In the Koster et al article (Lancet, Dec. 18, 1993, Vol.342, pages 1503-1506) it was stated that reduced levels of prothrombinand/or Factor X (<0.5 μ/ml) will increase the APC-SR. For this reasonthe test cannot be used for the evaluation of plasma's of patients onoral anticoagulant treatment. In a series of 98 samples a goodcorrelation was found between the APC-SR obtained with the test ofKoster et al (Lancet, Dec. 18, 1993, Vol. 342, pages 1503-1506) andthose obtained with the test developed by Chromogenix as described in WO93/10261. A reference range for the APC sensitivity ratio was derivedfrom the healthy control subjects. After logarithmic transformation ofthe data and exclusion of 10 subjects with values outside 3 standarddeviations (SD) of the mean, the lower limit of normal was 2.17(mean-1.96 SD). An inverse relation between the risk of thrombosis andthe degree of response was found. The 21% prevalence of a poor responseto APC among thrombosis patients and the odds ratio for thrombosis of6.6 led to the conclusion that a poor response to APC could beconsidered a common and strong risk factor for deep vein thrombosis. Itwas even speculated that subjects with APC sensitivity ratios around1.10 could be homozygous or double heterozygous, whereas subjects withAPC sensitivity ratios around 1.50 could be heterozygous for theabnormality. The prevalence of the abnormality was 5% among the healthycontrol subjects. Because the distribution of the APC-SR was clearlybimodal Koster et al believe that subjects really had abnormal responsesto APC rather than too low values within a normal range. The relationbetween risk of thrombosis and their response to APC seemed thereforenot to follow the model of a simple single gene defect. Because theabnormality was found to be so prevalent in healthy subjects it wasconsidered unlikely by Koster et al that the defect in itself issufficient to cause thrombosis as is true also for protein C deficiency(Ref. 15, Ref. 16). An additional causal factor seems to be required forthe development of thrombosis within a particular patient. These may beacquired factors and also as yet unknown genetic defects or variations.However once other causal factors are present poor APC response presentsa strong risk of thrombosis as witnessed by its six to sevenfoldincrease of relative risk. It was stated in the article that theunderlying defect of the poor response to APC remained to be clarifiedeven though a dominantly autosomally inherited deficiency of a cofactorto activated protein C had been postulated (Ref. 7). While a poorresponse to APC appears to be 5 to 10 times as frequent as deficienciesof protein C, protein S or antithrombin it confers an approximatelysimilar relative risk of thrombosis (Ref. 17 and 18) which according toKoster et al could make it worthwhile to test all patients with venousthrombosis for this abnormality.

In summary in the state of the art it was ascertained that a defect inthe protein C anticoagulant pathway is linked to a relatively high riskof thrombosis. The poor anticoagulant response to activated protein Chas been discussed in great detail, however the cause of the pooranticoagulant response to activated protein C remains unclear. A numberof theories have been postulated, however the only one that has beenaccepted is the presence of an unknown cofactor for APC which isapparently deficient in a patient exhibiting a poor anticoagulantresponse to activated protein C. The identity of the postulated cofactorfor APC is unknown. Furthermore current tests for detecting alteredresponse to APC cannot be used on test persons already usinganticoagulants.

DESCRIPTION OF THE INVENTION

Surprisingly the identity of the unidentified cofactor responsible for apoor anticoagulant protein C response has been found. It has beendiscovered that one of the mechanisms that had been rejected is in factresponsible for the defect in the protein C anticoagulant pathway in amajority of thrombophilic patients. The cause of the deficiency has beenlinked to the presence of a mutation in the nucleic acid materialencoding Factor V or Factor VIII which upon expression is correlated toa decrease in the degree of inactivation by APC of said Factor V and/orof Factor Va, (the product of activation of said Factor V) or of saidFactor VIII and/or of Factor VIIIa (the product of activation of saidFactor VIII). The deficiency is therefore not the result of a mutationin an as yet unidentified cofactor for APC, but is in fact due to adefect in Factor V or Factor VIII or more particularly in the activationproducts thereof.

As was already indicated in the state of the art the link between a riskof thrombosis and the presence of APC-resistence had already been madeand it had also been suggested that screening for such a deficiencywould in fact be extremely helpful in diagnosing patients with anincreased risk of thrombosis. As it is now known which factors carry thegenetic defect responsible it has also now become possible to actuallyscreen the population with methods other than the Chromogenix test fordetermining APC-resistance.

It has become possible to use DNA techniques or to use antibodies fordetermining the presence of mutant proteins when screening for themutated Factor V or Factor VIII associated with resistance to APC. Thesubject invention is therefore directed at a method for screening forthe presence of a genetic defect associated with thrombosis and/or pooranticoagulant response to activated protein C (APC), said genetic defectbeing indicative of an increased risk of thrombosis or said geneticdefect actually causing thrombosis in a patient, said method comprisingdetermination of the presence of a mutation in the nucleic acid materialencoding Factor V or factor VIII in a manner known per se, whichmutation upon expression of the nucleic acid material is correlated to adecrease in the degree of inactivation by APC of said Factor V and/or ofFactor Va (the product of activation of said Factor V) or of said FactorVIII and/or of Factor VIIIa (the product of activation of said FactorVIII) and/or comprising determination of a mutation present in theprotein Factor V and/or Factor Va and/or present in the protein FactorVIII and/or Factor VIIIa by analysis of said Factor V and/or Factor Vaor Factor VIII and/or Factor VIIIa or analysis of a proteolytic fragmentof said Factor V and/or said Factor Va and/or Factor VIII and/or FactorVIIIa in a manner known per se, said mutation being correlated to adecrease in the degree of inactivation by APC of said Factor V and/orsaid Factor Va and/or Factor VIII and/or Factor VIIIa. In particular themethod according to the invention is directed at a method wherein themutation in the nucleic acid sequence encoding the Factor V and/orFactor VIII is located at the position within the part of the nucleicacid sequence encoding a binding site or a cleavage site of APC onFactor V and/or Va and/or Factor VIII and/or VIIIa and results in FactorV and/or Factor Va and/or Factor VIII and/or Factor VIIIa poorlyinactivated by APC. There are known to be a number of binding andcleavage sites for APC in Factors V, VIII, Va and VIIIa. (see Table 1,refs. 34, 35, 36, 48, 49, 52 the article by Odegaard B & Mann K. G., inJ. Biol. Chem. 262, 11233-11238 (1987) and the abstract by Kalafatis M.,Haley P. E. & Mann K. G. in Blood 82, Suppl. 1, p. 58a, 1993).

TABLE 1 Cleavage and binding sites of activated protein C in Factor Vaand Factor VIIIa Cleavage or Human factor V(a) Bovine factor V(a)Cleavage or Human factor VIII or binding site sequence sequence Bindingsite VIIIa APC-cleavage R³⁰⁶-N³⁰⁷ (ref. 51) R³⁰⁶-N³⁰⁷ (ref. 48)APC-cleavage R³³⁶-M³³⁷ (ref. 49) APC-cleavage R⁵⁰⁶-G⁵⁰⁷ (ref. 51)R⁵⁰⁵-G⁵⁰⁶ (ref. 48,50) APC-cleavage R⁵⁶²-G⁵⁶³ (ref. 49) APC-cleavageR⁶⁷⁹-K⁶⁸⁰ R⁶⁶²-N⁶⁶³ (ref. 48,34) APC-cleavage R⁷⁴⁰-S⁷⁴¹ (ref. 49)APC-cleavage ? R¹⁷⁵²-R¹⁷⁵³ (ref. 48) APC-cleavage H²⁰⁰⁹AGMSTLFIV (ref.35) APC-cleavage ? R¹⁷⁵³-A¹⁷⁵⁴ (ref. 48) APC-cleavage R¹⁸⁶¹AGMQTPELI(ref. 35) R¹⁸⁵²AGMQTPELI (ref. 35) APC-cleavage K⁹⁹⁴ (ref. 52)

Binding sites are not always cleavage sites. However, it is quite clearthat any effect leading to reduced binding of APC to such a factor willalso have an effect on the APC resistance of such a factor, as generallyspeaking the factor must be bound by APC before it can subsequently becleaved by APC. The mutation affecting the binding and/or cleavage sitecan be present in the primary amino acid sequence of amino acids locatedat the binding site or can be due to a mutation elsewhere in themolecule resulting in a tertiary structure with a reduced affinity forAPC binding and/or cleavage. As a number of sites for APC binding and/orcleavage have been clarified it is obviously easiest to screen formutations at these locations rather than in the whole molecule. A numberof cleavage sites of APC are known to be located on the heavy chains ofthe Factors V, Va, VIII and VIIIa preferably the mutation to be detectedwill be located at a position within the part of the nucleic acidsequence encoding a cleavage site of APC on the heavy chain.

The activation of Factors V and VIII can occur by thrombin Factor Xa,and in the case of factor V also by some snake venoms. For “activated bya particular factor” a person skilled in the art can also read“activated via a particular factor”. The resulting activated factorsdiffer slightly due to the manner in which they have been activated. Itis therefore possible that a mutation resulting in reduced bindingand/or cleavage by APC of Factor Va activated by thrombin will notresult in reduced binding and/or cleavage by APC of Factor Va activatedby Xa and vice versa. Factor V has the following domain structureA1A2/BA3C1C2, Factor Va that has been activated by Xa has the structureA1A2/B′A3C1C2, whereas Factor Va that has been activated by thrombin hasthe structure A1A2/A3C1C2. It is probable that the tertiary structurediffers due to the variation in the structure of the light chains of thefactors when they have been activated in different manners. In theexample in this document it is illustrated that the particular mutationillustrated in fact only inhibited APC inactivation of Factor Va whenactivation was initiated via Xa and did not have an effect on APCinactivation of Factor Va that was activated via thrombin. Thisparticular mutation was located on the heavy chain of Factor V and istherefore present in both Factor V_(a) activated via thrombin and FactorVa activated via Xa but presumably does not exhibit the same effect inthe two forms of activated Factor V due to a differing tertiarystructure of the two forms of activated factors.

As in practice the inactivation by APC occurs on activated Factor V oractivated Factor VIII the subject method is preferably directed atdetection of a mutation resulting in a decrease of binding affinity forAPC and/or a reduction in cleavage by APC on Factors Va or VIIIa.Generally speaking mutations present in Factors Va and Factors VIIIawill also be present on the Factor V or Factor VIII from which theactivation product has been derived. Therefore analysis of the nucleicacid sequence encoding Factor V or Factor VIII will reveal mutationsthat can also be present in the activated Factors Va or VIIIa. Theanalysis according to the subject method can therefore be carried out atnucleic acid level e.g. at DNA and/or mRNA level of Factors V and VIIIand at protein level on any of factors Va, VIIIa, V and VIII, orfragments derived from these.

Factor Va DNA has been cloned from HepG2 cells (Ref. 20) and human fetalliver (Ref. 21 and 22). The complete Factor V amino acid sequence isknown (refs. 20, 23) also the organisation of the factor V gene has beenelucidated (Ref. 23). Shen et al (The Journal of Immunology, Vol. 150,2992-3001, No. 7, Apr. 1, 1993) have described how they found a cellularsource of human Factor V. They identified Factor V mRNA in humanlymphoid cells by using reverse transcription followed by the polymerasechain reaction (RT-PCR). The results from the PCR were confirmed byindependent cloning of Factor V cDNA from a T-cell cDNA library. Thesequence of the Factor V cDNA was virtually identical to hepatic FactorV mRNA. A limited span of mRNA encoding part of the connecting region ofthe Factor V protein was found to contain nucleotide polymorphisms basedon 6 nucleotide substitutions. Shen et al described that the amplifiedF7/F8 Factor V cDNA fragment, present in 14 independent clones derivedafter amplification, comprised 6 nucleotide base substitutions. 2Substitutions were of thymine to cytosine and from cytosine to thymineat positions 2209 and 2236 respectively that were silent mutations. Fourwere guanine to adenine base substitutions that also resulted in asilent mutation at position 2302 and also amino acid changes fromarginine to lysine at position 2573, from arginine to histidine atposition 2595 and from glutamic acid to lysine at position 2773. Thesededuced amino acid changes are described in the article as conservativesubstitutions that did not significantly affect Factor V function. Halfof the clones (7 of 14) in addition exhibited an adenine to guaninesubstitution at position 2290, another silent substitution whichabolished the EcoR1 site. None of these mutations have been associatedwith a decreased affinity for APC binding or cleavage.

In the article by Shen et al it is illustrated that Factor V mRNA can berecovered from human lymphoid cells in sufficient amounts to carry outpolymerase chain reactions for example. With this information a personskilled in the art will have no difficulty in retrieving nucleic acidfrom humans in a sufficient amount to carry out the method according tothe subject invention. In the Shen et al article a number ofoligonucleotides are presented which can be used in nucleic acidamplification of human Factor V nucleic acid.

Bruce Odegaard and Mann (The Journal of Biological Chemistry, Vol. 262,No. 23, August 15, pp. 11233-11238, 1987) described for both Factor Vand Va that cleavage of Factor V by thrombin results in a heavy chain (Dchain) of M_(r)=94.000 and a light chain (E chain) of M_(r)=74.000. Eachchain in itself is susceptible to proteolysis by activated protein C andby Factor Xa. Cleavage of the E chain by either activated protein C orFactor Xa yields two major fragments M_(r)=30.000 and M_(r)=48.000. Theyalso indicated that the activated protein C and Factor Xa cleave the Echain at the same position. The activated protein C cleavage of the Dchain yields two products M_(r)=70.000 and M_(r)=24.000. TheM_(r)=70.000 fragment has the same NH₂-terminal sequence as intact Dchain, the M_(r)=24.000 fragment does not. They illustrated that thecleavage of D chain by activated protein C was responsible for thepartial inactivation of Factor Va. Evidence that the inactivation ofFactor Va is associated with cleavage of its heavy chain, as the lightchain is cleaved at a slower rate, is also present in Refs. 10, 12, 12,14, and 48.

Odegaard and Mann also disclosed that there is a large deal ofsimilarity between Factor Va and Factor VIIIa. Factor VIIIa is anothercofactor of the clotting cascade whose activity is regulated byproteolysis. Factors VIIIa and Va have many similar structural andfunctional characteristics. Both are produced from a large procofactorthrough cleavage by thrombin or factor Xa, both are about the same sizeand consist of a heavy chain and a light chain. Both contribute greatlyto the activity of a proteolytic complex while not exhibitingproteolytic activity on their own, and both are inactivated by activatedprotein C. Underlying this commonality is also an apparent homology ofprimary structure (Ref. 24, 25 and 26). They also themselves identifiedfurther sequence homology between Factors V and VIII. In particular theystated there are clearly segments of sequence homology between bovineFactor V and human Factor VIII at positions in the Factor VIII moleculethat correspond reasonably well to the positions in the Factor Vmolecule at which cleavages occur. The effect illustrated by us forhuman Factor V (in example 1) can due to the equivalence of Factor V andFactor VIII in certain aspects also be expected concomitantly forFactors VIII and/or VIIIa. Odegaard and Mann also indicated that evenwhen no heavy chain remained after cleavage of Factor Va by APC thereremained residual cofactor activity. This indicated that theinactivation of Factor Va was a more complex event than simply thecleavage of a single bond, which makes the illustration of the examplesof this document that a mutation in an APC binding and/or cleavage siteof a Factor V molecule is in fact sufficient to cause a reduction inaffinity for inactivation by APC even more surprising. For Factor VIIIthe APC cleavage sites have been postulated at Arg 562, Arg 336 and Arg740 (Ref. 49) and the APC binding site in the A3 domain on residues2009-2018 (Refs. 35, 36) (see also Table 1).

The method according to the invention is directed at detecting one ormore mutations at one or more of the cleavage and/or binding sites forAPC of Factor V and/or Factor Va or at Factor VIII and/or VIIIa ateither nucleic acid or protein level or both. In particular the APCbinding and/or cleavage sites located on the heavy chain of the proteinor on nucleic acid encoding the heavy chain are considered relevant.

Kalafatis et al (Blood 82, Suppl. 1, p. 58A, 1993) illustrated thatmembrane bound human Factor Va was inactivated by activated protein Cafter cleavage of the heavy chain at Arg 506 and Arg 306. Theyillustrated that the cleavage pattern of the heavy chain of human FactorVa was dependent on the presence or absence of PCPS vesicles. In theabsence of a membrane surface or in the presence of phospholipidvesicles exclusively composed of PC, cleavage resulted in a fragmentcomprising residues 1-506 and a fragment starting with residue 507,which is further cleaved by APC at the COOH-terminus. In contrast, inthe presence of PCPS vesicles the complete loss of activity iscorrelated with the cleavage of the M_(r)=75.000 fragment and theappearance of M_(r)=40.000 and M_(r)=30.000 fragments. The M_(r)=30.000fragment corresponds to residues 307 to 506 demonstrating cleavage byAPC at Arg 306. No cleavage of the light chain of the cofactor isobserved in the presence as well as in the absence of PCPS vesiclesafter incubation with APC. Thus, a specific APC cleavage site is exposedwhen the cofactor is bound to PCPS. The presence of a membrane isessential for complete inactivation of human Factor Va by APC andcleavage at Arg 506 only partially inactivates the cofactor and cleavageat Arg 306 is anionic lipid dependent and is required for the completeinactivation of human Factor Va. Recently, similar data have beenpublished for the inactivation of bovine factor Va by APC (Ref. 48). Itis clear from the state of the art that there are thus at least twopotential cleavage sites in human Factor Va for APC. Kalafatis et alhave also detected an additional APC cleavage site at lysine 994 ofhuman factor V (ref 52). Therefore the method according to the inventionis directed at detecting mutations at one or more of these cleavagesites for APC in Factor V and/or Va at either nucleic acid or proteinlevel, or both. The cleavage sites for APC are located at Arg 506 andArg 306 on the heavy chain. A further site has been found to be presentat amino acid Arg 679 and Lys 994.

In view of the above the method according to the invention for screeningfor the presence of a genetic defect associated with thrombosis and/orpoor anticoagulant response to activated protein C (APC) said geneticdefect being indicative of an increase risk of thrombosis or saidgenetic defect actually causing thrombosis in a patient, said methodcomprising determination of the presence of a mutation in the nucleicacid material encoding Factor V in a manner known per se which mutationupon the expression of the nucleic acid material is correlated to adecrease in the degree of inactivation of APC of said Factor V and/or ofFactor Va is of particular interest when the Factor V has been derivedfrom Factor V activated by Xa.

In particular the method according to the invention is directed atdetermination of the mutation in a nucleic acid sequence encoding humanFactor V or Va with a mutated amino acid sequence comprising an alteredamino acid at a position corresponding to amino acid 506 of the sequenceof plasma Factor V (Ref. 21). In particular when the above-mentionedmutation is a mutation whereby the amino acid arginine has been replacedby the amino acid glutamine at amino acid 506 of the sequence of plasmaFactor V. This is in particular the case when the second nucleotide ofthe codon for the amino acid corresponding to amino acid 506, nucleotideG is mutated. In particular when the nucleotide G is mutated to A at theposition corresponding to the second nucleotide of the codon for theamino acid corresponding to amino acid 506 of the sequence of plasmaFactor V.

As is apparent from the Examples the subject invention is also directedat a method for determining whether a test person is homozygous orheterozygous for a mutation in Factor V and/or Factor Va or Factor VIIIand/or Factor VIIIa comprising carrying out a method known per se fordetermining whether a defect is present in anticoagulant response toAPC, subsequently followed by determination of a value of a parameterknown to be useful for diagnosis of the defect such as the (APTT+APC)over (APTT−APC) value and comparing the value obtained with a valueobtained in the same manner for a sample for a normal individual or froman individual known to be homozygotic or heterozygotic, therebyestablishing whether the test person is homozygotic or heterozygotic fora defect in anticoagulant response to APC, in combination with any knownmethod for determining the presence and optionally the nature of amutation in Factor V and/or Va or Factor VIII and/or Factor VIIIa inparticular in the embodiment illustrated in Example 1 and any equivalentembodiments of said Example for other mutations in Factors V, Va, VIIIand/or VIIIa resulting in altered anticoagulant response to APC, mostparticularly due to a mutation in an APC binding and/or cleavage site.

The method according to the invention can be accomplished by detectingthe mutation by carrying out a nucleic acid target amplificationreaction. Such target amplification reactions are well known to a personskilled in the art. It is required to use one or more primers specificto recognize and hybridize to stretches of nucleic acid adjacent to the5′ and 3′ end of the stretch of nucleic acid in which the mutation canbe located, said hybridisation being to a degree sufficient foramplification of the stretch of nucleic acid in which the mutation canbe located. The stringency of hybridisation required is also known to aperson skilled in the art of target amplification of nucleic acid. Thereare a number of target amplification reactions that are generallycarried out in the state of the art comprising NASBA (Nucleic AcidSequence Based Amplification) PCR (Polymerase Chain Reaction), LCR(Ligase Chain Reaction) and RCR (Repair Chain Reaction). For PCR targetamplification methods the Amplicor^(R) reaction kits are commerciallyavailable. It is also possible to use a primer sufficiently specific torecognize and hybridize to the stretch of nucleic acids in which themutation itself can be located. An alternative amplification methodcomprises branch chain amplification as commercially exploited by Chironwherein the probe rather than the target is amplified.

After amplification of the nucleic acid, analysis of the amplifiednucleic acid in a manner known per se for detecting the presence andoptionally the nature of the mutation is to be carried out in the methodaccording to the invention.

It is also possible to determine the mutation without amplification ofthe nucleic acid material. There are a number of techniques known to aperson skilled in the art that were used before the target amplificationreaction was developed for determining the presence of mutations onnucleic acid and these can all be used in various embodiments of themethod according to the invention. For example the mutation to bedetermined can be detected by a hybridisation reaction to at least onenucleic acid sequence sufficiently specific to hybridise to at leastpart of the nucleic acid sequence encoding the factor to be analysedwhen using normal to stringent hybridisation conditions e.g. blottingtechniques followed by analysis of the nucleic acid thus isolated in amanner known per se for detecting the presence and optionally the natureof the mutation.

The detection of the presence and optionally the nature of the mutationcan occur by subjecting the nucleic acid thus isolated to sequenceanalysis by using for example the Sanger sequence reaction to ascertainthe nucleic acid sequence and subsequently to compare the results ofthis sequencing with the sequence known for the non-mutated factor. Itis also possible to subject the nucleic acid sequence isolated to afurther hybridisation test. The further hybridisation test being carriedout with a stretch of nucleic acid material with a correspondingcomplementary sequence of sufficient length and specificity to at leasthybridize to a fragment of the nucleic acid material comprising themutation to detect the presence and optionally the nature of themutation. The first hybridisation step merely isolates nucleic acidencoding the factor whether this is mutated or not and the secondhybridisation step actually comprises hybridising the isolated sequenceto the complementary sequence of the actual mutated nucleic acidsequence one wishes to ascertain in order to determine the presence orabsence of said mutation on the isolated nucleic acid material. Thislatter hybridisation reaction should be carried out under stringentconditions for reliable results whilst the other hybridisation steps canbe carried out under normal to stringent conditions. Thus two classicalmethods for determining the presence of a mutation on a particularnucleic acid are hereby illustrated and it will be obvious to a personskilled in the art that a number of known techniques can be used. Invarious standard books for molecular biology such techniques are amplyillustrated for example in Sambrook, J., Fritsch, E. F., Maniatis, T.Molecular Cloning: a Laboratory Manual. (Cold Spring harbor LaboratoryPress, cold Spring Harbor, N.Y. 1989.

It is also possible to analyse the amplified nucleic acid materialobtained in the screening method according to the invention by usingsubsequent analysis tests using sequencing reactions or hybridisation toa corresponding complementary sequence of sufficient length andspecificity to at least hybridize to a fragment of the nucleic acidmaterial comprising the mutation to detect the presence and optionallythe nature of the mutation as was illustrated above for analysis ofisolated nucleic acid material that had not been subjected to anamplification reaction.

In particular for analysis of the presence of mutations in Factor V theisolated and/or amplified nucleic acid material can be subjected to ahybridisation test to a stretch of nucleic acid material selected fromsequences with sequence numbers 12 and 13 of the sequence listing. Forexample an extremely suitable primer or nucleic acid sequence forhybridisation comprises at least a part of intron 10 of the nucleic acidsequence encoding human Factor V or a derivative thereof capable ofhybridizing to said part of intron 10 under stringent conditions. Such aderivative will preferably be more than 90% homologous to thecorresponding part of intron 10. The nucleic acid sequence of humanFactor V is known and in sequence number 1 of the sequence listing thenucleic acid sequence encoding human Factor V is illustrated. Thesequence is derived from Ref. 21. Using the nucleic acid sequence forhybridisation comprising at least a part of intron 10 it is quite simpleto isolate and/or amplify and/or subsequently detect a mutation presentin nucleic acid encoding Factor V, in particular of nucleic acidencoding an APC binding and/or cleavage site. It is in particularsuitable to detect a mutation located on the heavy chain (see sequences10, 14). Further, primers of nucleic acid sequences for hybridisationand/or amplification purposes can be selected from sequences withsequence listing numbers 2-11 of the sequence listing. As is alreadyindicated above a number of other oligonucleotide primers are also knownfrom the state of the art. It is also possible to use these primers foramplification purposes or hybridisation reactions to isolate the nucleicacid encoding Factor V and/or Va. It lies within the reach of a personskilled in the art to select oligonucleotide sequences best suited toisolate and/or amplify and/or determine the presence and nature of themutation he is screening for in a method according to the invention asthe sequence encoding the normal factor is known, as is a sequence for amutated factor.

In the method according to the invention, in particular when the nucleicacid to be analysed has been subjected to target amplification, theisolated and/or amplified and/or hybridised nucleic acid material issubjected to sequence analysis and the sequence is then compared to thenucleic acid sequence of the corresponding non-mutated factor. It isalso possible to analyse the amplified or isolated and/or hybridisednucleic acid material through restriction fragment analysis. Inparticular for the mutation illustrated in the example with Factor Vthat is mutated, the enzyme that can be used in Mn1 I. Naturally therestriction enzyme one can use for a restriction fragment analysisdepends on the nature of the mutation to be detected and the locationthereof. Determination hereof lies within the reach of a person skilledin the art without involving further inventive step, merely routineexperimentation not placing an undue burden on a person skilled in theart.

As stated above the method according to the invention can also becarried out by analysing the protein rather than the nucleic acidsequence encoding the protein. In particular this is a useful embodimentof the invention when the mutation in the protein is located at aposition within the part of the amino acid sequence providing a bindingand/or a cleavage site of APC on Factor V or Va and results in Factor Vand/or Factor Va poorly inactivated by APC or is located on the part ofthe nucleic acid sequence providing a binding and/or a cleavage site ofAPC of Factor VIII or VIIIa and results in Factor VIII and/or FactorVIIIa poorly inactivated by APC.

As stated above the presence of a mutation within the part of the aminoacid sequence providing a cleavage site of APC on Factor V, Va, FactorVIII or VIIIa is a mutation that will quite clearly lead to an alteredresistance of the mutated factor to inactivation by APC and thereforedetermination of such a mutation is a preferred embodiment of the methodaccording to the invention. As already indicated in the state of theart, inactivation of Factor Va or Factor VIIIa generally ensues when APCcleaves the heavy chain of the factor. Therefore, detection of amutation in such a cleavage site resulting in an amendment of a degreeof cleavage of the mutated factor is a preferred embodiment of theinvention. When analysing the protein for a mutation it is not only theprimary amino acid sequence of the cleavage site itself that isrelevant, but also the tertiary structure of the protein can bedistorted due to a mutation somewhere in the primary sequence notimmediately associated with the binding and/or cleavage site. It is wellknown that mutations located quite a long way away from the actualbinding site or cleavage site of a protein can exhibit a large effect onthe tertiary structure of the protein thereby also abolishing orreducing binding to said protein in this instance binding by APC.Therefore the method according to the subject invention is not onlydirected at detection of mutations in the primary nucleic acid sequenceof the cleavage and/or binding sites for APC but also at detection ofmutations resulting in a mutated factor having an altered tertiarystructure resulting in reduced binding and/or cleavage of the factor byAPC.

As the Factors V and VIII can both be activated by different mechanismsand the resulting activated factors are known to differ both in tertiarystructure from the factors from which they have been derived it isapparent that the mutation in Factor V or Factor VIII could have noinfluence on the APC binding and/or cleavage sites of said molecules butcould have an effect on those of the activated factors or vice versa.Nevertheless the mutation in primary amino acid sequence responsible forthe altered binding and/or cleavage of the activated Factor V or VIIIwill also be present on Factor V or Factor VIII. When the detection ofthe mutated protein occurs by using a specific antibody it is possibleto use an antibody specifically directed against the activated factorcomprising the mutation for detection of the presence and optionally thenature of the mutation. Alternatively it is also possible toproteolytically cleave the protein to be analysed, thereby obtaininglinear or partially linear structures making it possible to useantibodies specific for the mutation in the primary amino acid sequenceof Factor V and/or Va or Factor VIII and/or VIIIa. Thus the detectionmethod of the mutation need not be restricted to analysis of theactivated factors but can in fact also occur on Factor V or Factor VIIIthat have not yet been activated.

If the mutation is present in a binding and/or cleavage site for APCthen treatment of Factor V, Va, VIII, VIIIa with APC followed byanalysis of the fragments in a manner known per se should revealdifferent fragments than when the Factor is normal.

For instance in the case of Factor V a mutation at amino acid 506prevents cleavage and/or binding of APC there. Treatment with activatedAPC will thus result in cleavage at sites 306, 679 and 994, providingone fragment of aa 307-aa 679 and three other fragments comprising asequence of 1-306, 680-994 and 995-terminus. The fragment of interestbeing 307-679. A normal Factor V will not comprise this fragment butwill comprise two other fragments i.e. aa 307-506 and aa 507-679 due tothe active cleavage site at aa 506. Thus detection of the aa 307-aa 679indicates the presence of a mutated APC site at amino acid 506.

An extremely elegant test could comprise subjecting the Factor V aftertreatment with APC to the presence of 2 antibodies. Such a treatmentwith APC naturally occurs during preparation of serum. One antibody inthe test being specific for a site of the protein upstream of amino acid506, said site being located downstream of aa 306, the most adjacentcleavage site upstream of aa 506. The second antibody being specific fora site of the protein downstream of amino acid 506, said site beinglocated upstream of aa 679, the most adjacent cleavage site for APCdownstream from aa 506. The test comprises detection of a fragmentdetected by both antibodies. Such a test can be a sandwich immunoaassay.Preferably one antibody will be immobilized or can be immobilized andthe other antibody will be provided with a detectable marker in a mannerknown per se for a person skilled in the art of immuno-assays. Use of anantibody specifically recognizing a part of the fragment 307-506 in sucha test falls within the scope of the invention. An antibody specificallyrecognizing a part of the fragment 507-679 as such and the use thereofin a test as just described falls within the scope of the invention.Preferably the antibodies will be monoclonal antibodies. A suitable testcan be carried out to detect a mutation in the APC cleavage site locatedat Arg 306 in an analogous manner using 2 antibodies, one specific for apart of fragment 1-306 and one specific for a part of fragment 307-506.Use of an antibody specifically recognizing a part of the fragment307-506 in such a test falls within the scope of the invention. Anantibody specifically recognizing a part of the fragment 1-306 as suchand the use thereof in a test as just described falls within the scopeof the invention. A test can also be carried out in an analogous mannerto that described above for detection of a mutation in the APC cleavagesite located at amino acid 679. for this mutation one antibody specificfor a part of the fragment 507-679 is required and one antibody specificfor a part of the fragment downstream of amino acid 680 is required. Useof an antibody specifically recognizing a part of the fragment 507-679in such a test falls within the scope of the invention. An antibodyspecifically recognizing a part of the fragment 507-569 and an antibodyspecifically recognizing a part of the fragment 570-994 as such and theuse of one or more of these antibodies in a test analogous to thatdescribed above falls within the scope of the invention. Analogously atest can be described for detection of a mutation at the APC cleavagesite at lysine 994. The fragments 994-terminus and 680-994 are therelevant fragments, as are the antibodies capable of recognising them.

In general terms the test for a mutation in Factor V, Va, VIII or VIIIacan comprise use of 2 antibodies in an immunoassay in a manner known perse to detect the presence or absence of a mutation decreasing orinhibiting cleavage by APC at a particular APC cleavage site, whereinone antibody recognizes a fragment upstream of said APC cleavage site,the second antibody recognizes a fragment downstream of said APCcleavage site, and no other APC cleavage sites are located between thepart of the Factor either antibody recognizes and the particular APCcleavage site for which the presence or absence of a mutation has to bedetermined. The various embodiments following from this principal ofmutation detection will be obvious to a person skilled in the art ofimmunoassays. It will thus be obvious for example that one or moreadditional proteases can be used in combination with APC, said APChaving to be added or already being present in the sample depending onwhich type of sample is used. The additional protease or protease beingselected such that the absence of the active APC cleavage site to bedetected on the Factor results in the binding of both antibodies to theproteolytic fragment comprising the inactive APC cleavage site, whereasthe presence of the active APC cleavage site to be detected results inproteolytic fragments such that the antibodies cannot both bind to aproteolytic fragment. This is most simply arrived at the selection ofone or more protease resulting in cleavage of the Factor upstream anddownstream of the APC cleavage site to be analysed and one of the twoantibodies recognising a part of the fragment upstream of the APCcleavage site to be determined and downstream of the location theprotease cleaves upstream of the APC cleavage site and the other of thetwo antibodies recognising a part of the fragment downstream of the APCcleavage site to be determined and upstream of the location the proteasecleaves downstream of the APC cleavage site and the protease orproteases cleaving the Factor such that their cleavage sites are locatedbetween the APC cleavage site to be determined and the adjacent APCcleavage site as present on a non mutated Factor. In yet a furtherembodiment one can apply in lieu of APC a protease capable of cleavageof the mutated APC cleavage site but not of the non mutated APC cleavagesite or vice versa. Once the nature of the mutation to be determined isascertained it is a matter of routine experimentation for a personskilled in the art to screen the recognition sites known for proteasesfor a suitable protease.

A further possibility for detection of the mutation lies in the oldertechnique of amino acid sequence analysis. Once the amino acid sequenceof the non-mutated factor is known it is quite simple to determine theamino acid sequence of the factor to be analysed and compared thatsequence to the known sequence of the corresponding non-mutated factor.However, using antibodies is a simple and efficient way to analyseproteins for the presence of mutations, for example using ELISA's orRIA's or a variety of other immunological tests known to a personskilled in the art.

It is also possible that the activated forms of Factor V and Factor VIIIonly exhibit a reduction in APC binding and/or cleavage when activatedby one particular mechanism. This is illustrated in Example 1 for FactorV, wherein the activated form that has been activated using thrombindoes not exhibit any altered binding and/or cleavage capacity for APCwhereas the activated form that has been activated by Factor Xa doesexhibit a reduction in the binding and/or cleavage by APC. However, asstated above, it is of course possible to detect the presence of themutation on either Factor V, Factor Va activated by thrombin or FactorVa activated by Xa, regardless of whether the effect of the mutationonly occurs in one of the activated forms.

As is illustrated in Example 1 a specific mutation in Factor V has beendiscovered that is representative for a very large percentage ofpatients exhibiting thrombophilia without the cause thereof having beenpreviously determined. This concerned a mutation of the amino acid at aposition corresponding to amino acid 506 of the amino acid sequence ofplasma Factor V (as disclosed in Ref. 21) and therefore a method whereinthe mutation of amino acid 506 can be determined forms a preferredembodiment of the invention. In general a method wherein the mutation tobe detected comprises an alteration of the arginine amino acid locatedin an APC cleavage site, in particular on a heavy chain of Factor V(a)and/or Factor VIII(a) is an embodiment of the subject method that can besuitably carried out.

For detecting the mutation one can use a specific antibody capable ofbinding to the mutant protein V and/or Va or of binding to a linearproteolytic fragment of the mutant protein Factor V and/or Va, saidantibody having a lower binding affinity for the non-mutated protein orfor the corresponding proteolytic fragment of the non-mutated protein.The method with antibodies can also be used for protein Factor VIIIand/or VIIIa and linear proteolytic fragments of said mutant proteinFactor VIII and/or VIIIa.

It is also possible as an alternative to use an antibody capable ofbinding to a protein Factor V and/or Va or a protein Factor VIII and/orFactor VIIIa, said protein not exhibiting a decrease in the degree ofinactivation by APC, said antibody having a lower binding affinity forthe corresponding factor and/or for the proteolytic fragment thereofcomprising a mutation resulting in the mutated protein exhibiting adecrease in the degree of inactivation by APC. In this instance a testcan be developed whereby non-binding of the antibody to the isolatedprotein or proteolytic fragment is illustrative of the presence of amutation. The invention is not only directed at methods using antibodiesas described above but is also directed at the antibodies themselves.

In the method according to the invention it is possible to first screena sample for an altered coagulation time upon addition of APC incomparison to that of a normal plasma standard, followed by analysis ofthe nucleic acid sequence encoding Factor V or Factor VIII and/or ofamino acid sequence of Factor V, Va, VIII or VIIIa and/or analysis ofFactor V, Va, VIII or VIIIa itself once the sample is diagnosed asexhibiting altered APC resistance in comparison to the standard. It isalso possible to immediately subject a sample to analysis for thepresence of a specific mutation, for example for the mutation in FactorV illustrated in the following Examples. The methods to be used willdepend on the circumstances of the case and also the objective of thetest. For example when screening a large population the cheapest methodto be used will be preferred. In some instances the mutation to bedetected will be difficult to determine using antibodies and then use ofnucleic acid sequences or restriction fragment analysis can bepreferred. Also if the enzyme to be used for a restriction fragment testis inexpensive then carrying out such a test is very simple and cheap tocarry out and will obviously be suitable. The invention is thereforealso directed at use of a test for determining whether a sample exhibitsaltered binding and/or cleavage by APC in comparison to a samplecomprising normal Factor V and/or Factor VIII and/or Factor VIII_(a)and/or Factor Va, followed by further analysis of the mutation causingthis alteration in a manner elucidated above.

Another aspect of the invention lies in the detection of a mutation inFactor V, Va, VIII or VIIIa being homozygous or heterozygous in the testperson. This can be carried out using the test protocol as described byKoster et al in Lancet, Dec. 18, 1993, Vol. 34 for determining whether atest person exhibits APC resistance. Basically Koster describes use of50 μl undiluted plasma incubated with 50 μl APTT reagent (Cephotest®,batch 103029) for 360 seconds at 37° C. before clot formation wasstarted either with 50 μl of a reagent containing 33 mM CaCl₂, 25 mMTris (pH 7.5), 50 mM NaCl and 0.05% ovalbumin (APTT-APC) or with 50 μlof the same reagent also containing 2.0 μg/ml human APC and 0.6%glycerol (APTT+APC). He expressed his results as APC sensitivity ratio's(APC-SR) defined as the ratio of APTT (+APC) and APTT (−APC). Underthese conditions the APC-SR is achieved for normal plasma. Reducedlevels of prothrombin and/or Factor X (<0.5 U/ml) will increase theAPC-SR. This method therefore cannot be used for evaluation of patientson oral anticoagulant treatment. Koster further stated that with thistest he found a good correlation between his results and those obtainedusing the Chromogenix assay (Pearson correlation coefficient 0.54)discussed in the introduction. Surprisingly we discovered that theKoster test when applied for assessing whether a subject is heterozygousor homozygous for mutation in Factor V detects abnormals a lot betterthan the Chromogenix test which misses approximately half theheterozygotes. FIG. 14 is the Koster test and FIG. 11 is the Chromogenixtest. We carried out tests twice during the Koster method and theChromogenix test as commercially available on samples from a randomselection of individuals genotyped as 1691 GG (normals) or 1691 AG(heterozygotes). In the Chromogenix test there was a great deal ofoverlap between the sensitivity ratios obtained in normals andheterozygotes, so that more than 50% of the heterozygotes could not beidentified as APC resistant with the Chromogenix test. We therefore havefound an additional test suitable for determining whether a test personis abnormal or normal for Factor V mutation resulting in APC resistance.Whereas previously the Koster test was merely postulated to be useful todetermine whether a test person is normal or abnormal with regard to APCresistance in general we have now discovered it in fact detects Factor Vmutation leading to APC resistance and in addition it does to a muchhigher degree of reliability than the Chromogenix test can. A value ofless than 0.84 is abnormal in our test and no overlap occurs betweennormal subjects and heterozygotes. This is a significant improvementover the Chromogenix test. We believe the improvement is due to the useof a different activator and more importantly the use of a differentcalcium concentration than in the Chromogenix test. The improved testcomprises applying a calcium concentration of more than 25 mM CaCl₂ inthe sample. Preferably less than 45 mM, more preferably 30-40 mM, inparticular 31-35 mM. This higher concentration probably neutralizescitrate in the sample to a better degree than in the Chromogenixformula. A further improvement lies in the use of Cephotest reagent asactivator. The method is further analogous to the Chromogenix test andmust be considered a considerable improvement thereof. When valuesobtained for the APC sensitivity ration have been normalised (seeExample 1) a value below 0.84 is indicative of abnormally and a valueabove 0.084 is indicative of normally regarding APC resistance, inparticular related to Factor V mutation. For homozygotes deter,omatopm avalue below 0.50 must be registered, with heterozygotes exhibitingvalues between 0.50 and 0.70. This improved method however is notapplicable on patients that have been subjected to treatment withanticoagulant.

In the example the mutation that was detected was the G→A mutation atthe codon for amino acid 506 of Factor V. The frequency of occurrence ofthe mutation and the associated high risk of thrombosis means thatdetermination whether a test person is homozygous or heterozygous isextremely relevant when assessing the risks for parents for passing onthe mutated factor to their progency. In Example 2 we illustrate thatthe presence of a mutation in Factor V, in particular the G→A mutationin fact is a risk factor for developing thrombosis. Moreover thepreliminary observation that 6% of the patients with a first myocardialinfarction is a carrier of the 1691 G-A mutation, might indicate thatthis mutation is also a mild risk factor for arterial thrombosis(relative risk 1,5-2,0). The timely detection of an increased risk forheart attack can lead to a person adjusting like style and takingprecautions to prevent such an event. The importance regarding venousthrombosis has already been discussed in the introduction.

The invention is also directed at kits comprising the elements necessaryfor carrying out the method according to the invention in all theembodiments illustrated. This comprises for example test kits comprisingone or more of the specific antibodies described above, in particularthe parts of antibodies described recognizing sites between APC cleavageand/or binding sites and/or comprising one or more probes or primers orpairs of primers for target amplification reactions and/or hybridisationreactions as described above. Specifically the invention is directed ata kit comprising a primer or primers for amplifying the nucleic acidsequence comprising the mutation of the nucleic acid sequence coding foramino acid 506 of Factor V and/or Factor Va. The kit can compriseprimers and/or antibodies for the detection of one particular mutationor for a number of mutations. Preferably the kit will comprise thecomponents necessary to detect the major mutations leading to a decreaseand/or abolition of binding and/or cleavage by APC that are prevalent inspecific populations.

EXAMLE 1

Recently a poor anticoagulant response to APC (“APC-resistance” (Ref.2)) was found in the plasma of 21% of unselected consecutive patientswith thrombosis (Lancet, Dec. 18, 1993, Vol. 342, pp. 1503-1506, T.Koster et al) and about 50% of selected patients with a personal orfamily history of thrombosis (Ref. 8 and Blood, Vol. 82, No. 7 (October1), 1993: pp. 1989-1993, J. H. Griffin et al). Here we demonstrate thatthe phenotype of APC-resistance is associated with heterozygosity orhomozygosity for a single point mutation in the factor V gene (1691,G−A) which predicts the synthesis of a factor V molecule −FV (Q506) orFV Leiden- which is resistance to inactivation by APC. The allelicfrequency of the mutation in the Dutch population is about 2% and is atleast ten-fold higher than that of all known genetic risk factors forthrombosis (protein C- (Ref. 17), protein S- (Ref. 30), antithrombin III(Ref. 31) deficiency) together (Ref. 32).

Our previous finding that 5% of apparently healthy individuals have apoor anticoagulant response to APC and that this APC-resistance isassociated with a seven-fold increase in the risk for deep veinthrombosis (Lancet, Dec. 18, 1993, Vol. 342, pp. 1503-1506, T. Koster etal), prompted us to investigate the molecular basis of this phenotype.

The responsiveness of plasma to APC is measured as the ratio of twoActivated Partial Thromboplastin Times (APTT), one measured in thepresence of APC and one int its (Lancet, Dec. 18, 1993, Vol. 342, pp.1503-1506 T. Koster et al; Blood, Vol. 82, No. 7 (October 1), 1993: pp.1989-1993, J. H. Griffin et al and Ref. 2). For reasons ofstandarisation this ratio (APC-Sensitivity Ratio or APC-SR) isnormalized to the ratio obtained with a reference plasma (n-APC-SR).Resistance to APC is defined by a n-APC-SR<0.84 (1.96 SD below the meann-APC-SR in 100 healthy controls, after the removal of outliers).

Analysis of the parentships of 14 unrelated APC-resistant patients ledto the concept of a familial form of APC-resistance (or APC-cofactor IIdeficiency (Ref. 2)) where homozygotes and heterozygotes can beidentified on the basis of the n-APC-SR (see legend of FIG. 1). Furthersupport for this concept was obtained from mixing experiments (FIG. 1);addition of one volume of normal plasma to one volume of the plasma of apatient classified as homozygous APC-cofactor II deficient (n-APC-R0.38) results in a n-APC-SR of 0.57. This is identical to the ratiofound in plasma of patients classified as heterozygotes for thedeficiency (mean n-APC-SR 0.58). Mixing the plasma of four unrelatedpatients, classified as homozygous APC-cofactor II deficient (meann-APC-SR 0.40) did not reveal any correction of the ratio, indicatingthat in all four patients the same plasma protein was missing ordefective (see also Ref. 2 and Blood, Vol. 82, No. 7 (October 1), 1993:pp. 1989-1993, J. H. Griffin et al).

To investigate the possibility that APC-cofactor II activity is afunctional feature of one of the known blood coagulation proteins,APC-cofactor II levels were measured in a series of plasmas deficient ofone single protein (FIG. 2). All these plasmas contained normalAPC-cofactor II levels (60-155%) except factor V deficient plasma (<5%).Addition of different amounts of isolated human factor V to factor Vdeficient plasma introduced both factor V coagulant activity andAPC-cofactor II activity.

Independent support for the candidature of factor V as APC-cofactor IIwas obtained from linkage studies in a large family with APC-resistance(FIG. 3).

The human locus for the factor V gene (F5) has been mapped to chromosome1 (1q21-25) (Ref. 33). There are no reports of convenient (PCR-able)polymorphic F5 markers. However, variations in published factor V cDNAand genomic sequences (Refs. 20-23) and The Journal of Immunology, Vol.150, 2992-3001, No. 7, Apr. 1, 1993, N. L. L. Shen et al) aided us toidentify two new polymorphisms in the factor V gene. Unfortunately, bothwere not informative in the APC-resistant family. Therefore we testedthe segregation of microsatellite markers for several loci in the1q21-25 region (see FIG. 4) in this family. The table in FIG. 5 showsthe pairwise lodscores for linkage between these markers and thephenotype of APC-resistance. Significantly positive results wereobtained only for locus D1S61 (Zmax 7.27 at θ=0.00), which is locatedwithin 4 cM from the F5 locus.

At this point we believed to have sufficient indications thatAPC-resistance is associated with a detect in the factor V gene to startthe search for the relevant mutation(s). We focussed our investigationson two regions in factor V, that contain the putative APC binding site(residues 1865-1874) (Refs. 35,36) and the putative APC cleavage site(Arg-506) (Ref. 21 and The Journal of Biological Chemistry, Vol. 262,No. 23, August 15, pp. 11233-11238, 1987, Bruce Odegaard and KennethMann), respectively.

As a first approach, ectopic transcripts of the factor V gene, isolatedfrom peripheral blood lymphocytes, were used for first-strand cDNAsynthesis and subsequent amplification of the two regions coding for theAPC binding and cleavage site. Direct sequencing of the PCR-fragmentsrevealed that two unrelated patients, classified as homozygous deficientof APC-cofactor II, were both homozygous for a 1691, G→A transition(FIG. 6). This mutation predicts the replacement of Arg-506 (CGA) by Gln(CAA) (FV(Q506) or FV Leiden). No other sequence abnormalities wereobserved in 225 bp surrounding 1691 A and in 275 bp around the regioncoding for the putative APC binding site (FIG. 7).

If cleavage after Arg-506 is instrumental for the inactivation of humanfactor Va by APC, one would predict that introduction of a Gln inposition 506 will prevent the inhibitory cleavage. During thecoagulation process plasma factor V is initially activated by factor Xa(formation of 105/220 kDa heterodimer (ref. 37)) and next furtherprocessed by thrombin (formation of 105/74 kDa heterodimer (Ref. 38))(Ref. 39). Interestingly, we found that the replacement of Arg-506 byGln only prevents the inactivation of the Xa-activated form of factor Vby APC (FIG. 8) but not that of the thrombin-activated form (data notshown).

The observation that two unrelated APC-resistant patients werehomozygous for the same mutation, suggested that this alteration ispresent in the majority of APC-resistant patients. To investigate thispossibility a test was designed for the screening of genomic DNA for thepresence of the 1691 G→A transition. Because the mutation is located inexon 10, 11 nt 51 from the start of intron 10 and only the first 8nucleotides of intron 10 have been published (Ref. 23), more intron 10sequence was generated by hemi-nested reverse PCR (Ref. 40) (see alsosequence 14). From this information primers were designed for theamplification of two overlapping genomic fragments that could be usedfor genotyping.

Digestion of the 267 bp fragment with Mnl I was used to demonstrate thepresence of a normal (1691 G) or mutated allele, while hybridisation ofthe 222 bp fragment with oligonucleotides specific for the normal ormutated allele was used for the positive identification of 1691 A. Usingthis approach we first studied all the members of this pedigree fromFIG. 3. Complete cosegregation of heterozygosity for the 1691 G→Atransition with APC resistance (n-APC-SR<0.84) was demonstrated as shownin FIG. 10 for a part of the pedigree. In addition, 4 patients (II.6,II.8, II.14, III.22) for whom no n-APC-SR was available because theywere treated with oral anticoagulants, were found to be heterozygous.

In a previous study of 301 consecutive patients with a first objectivelyconfirmed episode of deep vein thrombosis and 301 age and sex matchedpopulation controls. 64 APC-resistant thrombosis patients had beenidentified (Lancet, Dec. 18, 1993, Vol. 342, pp. 1503-1506, T. Koster etal). These 64 patients and their 64 controls were screened for thepresence of the G→A transition. From the 128 individuals 70 had an-APC-SR<0.84 (64 patients and 6 controls). Fifty six of these carriedthe mutation (53 patients and 3 controls), six of the patients in bothalleles (mean n-APC-SR 0.43; range 0.41-0.44) and the other 50 in oneallele (mean n-APC-SR 0.57; range 0.50-0.67). The remaining 14APC-resistant individuals did not carry the mutation, and had only amarginally reduced n-APC-SR (mean n-APC-SR 0.78; range 0.70-0.83). All58 non APC-resistant individuals did not carry the mutation (meann-APC-SR 0.99; range 0.83-1.19). Further none of 100 consecutivethrombosis patients with a n-APC-SR>0.84 was carrier of the mutation,while -as expected-3 of their 100 matched controls were. These 3 (meann-APC-SR of 0.57, 0.58 and 0.59) were the only controls with an-APC-SR<0.84.

Our data demonstrate that 80% of the individuals with a n-APC-SR<0.84and 100% of those with a n-APC-SR<0.70 are heterozygotes or homozygotesfor the 1691, G→A transition and that vice versa all carriers of themutation have a n-APC-SR <0.7. The relatively high frequency of themutated allele in the Dutch population (about 2%) combined with ourprevious finding (Lancet, Dec. 18, 1993, Vol. 342, T. Koster et al) thatAPC resistance is a common and strong risk factor for deep veinthrombosis, makes this hereditary factor V defect the most commonhereditary blood coagulation disorder sofar.

FIGS. 1 and 2.

Measurement of APC-cofactor II levels in plasma

FIG. 1. Calibration curve for the assay of APC-cofactor II activity inplasma.

APC-cofactor II refers to the hypothetical new cofactor of APC (Ref. 2)which is missing or defective in individuals with APC-resistance.n-APC-SRs were measured in dilutions of normal plasma (100% APC-cofactorII) in plasma of a patient homozygous deficient of APC-cofactor II (0%APC-cofactor II). The curve in FIG. 1 is the result of nine differentexperiments. The classification homozygous or heterozygous deficient ofAPC-cofactor II is based on the results of parentship analysis for 14probands with APC-resistance (n-APC-SR<0.84). For 2 probands (n-APC-SR0.38/0.41) both parents were APC-resistant (mean n-APC-SR 0.55); for 11probands (mean n-APC-SR 0.57) one of the parents was APC resistant (meann-APC-sR 0.59) while the other was (mean n-APC-SR 0.96); for one proband(n-APC-SR 0.74) both parents were not affected (n-APC-SR 0.96/0.99). Wepropose that individuals can be classified a homozygotes orheterozygotes for APC-cofactor II deficiency on the basis of theirn-APC-SR (homozygotes: mean 0.40; n=2; heterozygotes: mean 0.58, range0.51-0.67, n=26).

FIG. 2. APC-cofactor II activity levels in plasmas deficient (<5%) of asingle coagulation factor.

Plasmas were either from patients with a congenital deficiency (a,g,f,m,g,r,s,t) or prepared by immunodepletion (b,c,d,e,j,h,i, k,l,p).Plasmas were deficient of factor II (a), factor VII (b), factor IX (c),factor X (d), factor XI (e), factor XII (j), factor XIII (g), protein C(1), protein S (i), β2-glycoprotein (j), antithrombin (k), factor V(l,m), factor VIII (p,q) or von Willebrand factor (r,s,t). Factor Vdeficient plasma (m) was supplemented with two differentconcentrations—54% (n) and 90% (o)—of purified human factor V (Serbio,Gennevilliers, France), dialyzed against 20 mM sodium citrate, 150 mMNaCl, 4 mM CaCl₂ and tested for APC-cofactor II activity.

Methods:

The APC-SR was calculated from the results of two APTT measurements, onein the presence of APC and one in its absence, exactly as previouslydescribed. (Lancet, Dec. 18, 1993, Vol. 342, T. Koster et al) Then-APC-SR was calculated by dividing the APC-SR for the test sample bythe APC-SR for pooled normal plasma. APC-cofactor II activity wasmeasured by reading the n-APC-SR for two different dilutions (1:1, 3:4)of the test plasma in APC-cofactor II deficient plasma on a calibrationcurve as shown in FIG. 1.

FIGS. 3-5. Linkage analysis in a family with APC-resistance

FIG. 3. Pedigree of a family with APC-resistance (or APC-cofactor IIdeficiency).

, ▪, individuals with n-APC-SR<0.94 (mean0.65; range 0.59-0.71, n=13);

, □, individual with n-APC-SR>0.84 (mean 1.03; range 0.87-1.29;

n=20); , patients treated with oral anticoagulants (measurement ofn-APC-SR in these patients is not meaningful) ⊙, , individuals that werenot treated.

FIG. 4. Integrated genetic linkage map of the q21-25 region ofchromosome 1.

The relative positions of the loci AP0A2, D1S104, D1S61, AT3, LAMB andF13B were derived from the NIH/CEPH Collaborative Mapping Group linkagemap (Ref. 41). The genetic distance between adjacent loci is given incM. The F5 locus was placed on this map within 4 cM of the D1S61 locusby studying the segregation of markers for the F5 and D1S61 loci in 3CEPH families informative for both markers (in 55 meioses norecombination between these two loci was observed Z_(max) 16.6 atθ=0.00).

FIG. 5. Pairwise lodscores of APC resistance with chromosome 1 markers.

All available individuals of the pedigree in FIG. 3 were analyzed.Oligonucleotide sequences for markers for the loci ApoA2, D1S104, D1S61,LAMB and F13B are available from the Genome Data Bank. The primers wereobtained from the Dutch primer base. Three different polymorphic markersfor the AT3 locus were not informative in this family. Two point linkageanalysis was performed using the MLINK programs from the LINKAGE packageversion 5.3, which was obtained from Dr J. Ott. Sex averaged lodscoresare shown.

Methods

Microsatellite markers for ApoA2, D1S104, D1S61, LAMB and F13B wereamplified by PCR. Conditions: 50 mM NaCl, 10 mM Tris-HCl (pH 9.6), 10 mMMgCl₂, 0.01% BSA, 200 μM dGTP, dATP and dTTP, 20 μM dCTP, 0.7 μCi α³²pdCTP, 0.43 U Taq polymerase (Cetus, Emeryville, Calif., USA), 50 ng ofeach primer and 30 mg genomic DNA. 27 cycles were run at 94° C. (1′),55° C. (2′), 72° C. (1′) with a final elongation step of 10 min. PCRproducts were separated on a 6% denaturing polyacrylamide sequence gel,after which gels were dried and exposed to X-ray film.

F5 polymorphisms: A 636 bp fragment from exon 13 of the factor V gene(Ref. 23) was amplified by PCR using the primers number 2 (PR-776, nt2253-2272 (Ref. 21)) and number 3 (PR-768, nt 2870-2899 (Ref. 21)) ofthe Sequence Listing. For PCR conditions see legend FIGS. 10+11.Restriction with Hinf I detects a C/T dimorphism at nt 2298 (C: 0.68; T:0.32) and a rare A/G dimorphism at nt 2411 (A:0.98,; G:0.02). None ofthese markers was informative in the pedigree of FIG. 3.

FIGS. 6-8. Identification of the factor V gene mutation in a patienthomozygous deficient of APC-cofactor II

FIG. 6. Autoradiogram showing the nucleotide substitution in a patientclassified as homozygous deficient of APC-cofactor II.

Part of the nucleotide sequence of the non-coding strand of a cDNAPCR-fragment (coding for aminoacids 417 through 572 in human factor V(Ref. 21)) is shown for one patient (P) and one non APC-resistantcontrol (C). Arrows indicate the location of the 1691, G→A transition,which predicts the replacement of Arg 506 by Gln.

FIG. 7. Schematic representation of the factor V molecule.

Human factor V is a 330 kDa glycoprotein which contains several types ofinternal repeats. (Ref. 21) Activation by factor Xa results in theformation of a 105/220 kDa hetero-dimer (A₁A₂/B′A₃C₁C₂) (Ref. 38), whileactivation by thrombin results in the formation of a 105/74 kDaheterodimer (A₁A₂/A₃C₁C₂) (Ref. 37). APC binds to the A3 domain offactor Va (Ref. 35,36) and inbibits bovine factor Va by cleavage in theA2 domain after Arg-505 (The Journal of Biological Chemistry, Vol. 262,No. 23, August 15, pp. 11233-11238, 1987, Bruce Odegard and KennethMann).

The amino acid sequences surround the (putative (Ref. 43) APC cleavagesite in human (Arg-506) and bovine (Arg-505) factor Va are shown. In theACP-resistant patient Arg-506 has been replaced by Gln.

FIG. 8. Resistance of factor Xa-activated factor V(Q506) to inactivationby APC.

Al(OH)₃-adsorbed and fibrinogen depleted plasma (2 hr 37° C.; 0.3 U ml⁻¹Arvin) containing either factor V(R506) or factor V(Q506) was treatedwith factor Xa (2 nM) in the presence of 20 mM CaCl₂ and 20 μM PS/PC(25/75). After 8 min, when factor V activation was complete, 1.9 mM APCor buffer were added. At different time intervals 10 μl sample wasdiluted 1/100 in step buffer (50 mM Tris-HCl, pH 7.9, 180 mM NaCl, 0.5mg ml⁻¹ OVA, 5 mM CaCl₂) and directly assayed for factor Va activityusing the method described by Pieters et al (Ref. 44) The factor Vaactivity measured after complete activation of 0.70 U m⁻¹ FV(R506) (0.64μM thrombin min⁻¹) or 0.49 U ml⁻¹ FV(Q506) (0.20 μM thrombin min⁻¹) isarbitrary put at 100%; 0, −APC; , +APC.

Methods

cDNA synthesis: RNA was isolated (Ref. 45) from the lymphocyte fractionof 10 ml citrated blood of consenting patients and nonAPC-resistantcontrols. 1 μg of RNA was used as template for first strand cDNAsynthesis in the presence of mixed random hexamers using the superscriptkit (BRL, Bethesda, Md., U.S.A.). Amplification of cDNA fragments. Theprimers Sequence 4 (PR-764, nt 1421-1440 (Ref. 21)) and Sequence 5(PR-856, nt 1867-1891 (Ref. 21)) amplify the region coding for residues417 through 572 which contains the putative APC cleavage site; theprimers Sequence 6 (PR-849, nt 5608-5627 (Ref. 21)) and Sequence 7(PR-848, nt 6040-6063 (Ref. 21) amplify the region coding for amino acidresidues 1812 through 1963, which contains the APC binding region. PCRconditions were as described in the legend of FIGS. 9+10. PCR fragmentswere purified on ultra low gelling temperature agarose and directlysequenced as described before (Ref. 42) using the same primers as in thePCR reaction. One additional primer was synthesized to aid in sequencingof the APC-binding region:

Sequence 8(PR-847, nt 5905-5927 (Ref. 21)).

FIGS. 9+10. Association of APC-resistance with the presence of a 1691 Aallele of factor V

FIG. 9. Cosegregation of 1691 A with APC resistance.

The upper part gives the position of the individuals in the pedigree(FIG. 3) and the n-APC-SR, if available (II6 is on oral anticoagulanttreatment). The middle part shows the result of the Mnl I digestion ofthe 267 bp PCR fragment. The lower part shows the results of the dotblot hybridisation of the 222 bp fragment with the biotinylatedoligonucleotide specific for the 1691A allele (PR 1005).

FIG. 10. Dot blot hybridisation of the 222 bp PCR fragments of 64thombosis patients with a n-APC-SR<0.84 and their 64 matched controlswith the biotinylated oligonucleotide specific for the 1691 A allele (PR1005).

All patients (P) and controls (C) gave their informed consent. Slashesdenote positions of failed PCR reactions in this experiment.

Methods

Amplification of genomic fragments containing 1691 G/A. For Mnl-Idigestion a 267 bp fragment was amplified using as 5′ primer Sequence9(PR-6967;nt 1581-1602 (Ref. 21)) and as 3′ primer primers Sequence10(PR-990; nt 127 to −146 in intron 10). For dot blot hybridisation a222 bp fragment was amplified using as 5′ primer Sequence 11 (PR-6966,nt 1626-1647 (Ref. 21)) and as 3′ primer PR-990 (Sequence 10).Conditions: 125 μl of a mixture containing 54 mM Tris-HCl (pH 8.8). 5.4mM MgCl₂, 5.4 μM EDTA, 13.3 mM (NH₄)₂SO₄, 8% DMSO, 8 mMβ-mercaptoethanol, 0.4 mg ml⁻¹ BSA, 0.8 mM of each nucleosidetriphosphate, 400 ng of each primer, 200-500 ng DNA and 2U Taqpolymerase (Cetus, Emeryville, Calif. USA), was subjected to 36 cyclesof 91° C. (40″), 55° C. (40″) and 71° C., (2′). The 267 bp fragment(7-10 μl) was digested with 0.4 U Mnl I (Biolabs, Cambridge, Mass.,USA): the 1691 G fragment will give fragments of 67, 37 and 163 bp,while the 1691 A fragment will give fragments of 67 and 200 bp. The 222bp fragment (about 100 ng) was used for dot blot hybridisation withbiotinylated sequence specific oligonucleotide Sequence 12 (PR-1006; nt1682-1699 (Ref. 21)) for detection of 1691 G and Sequence 13 (PR-1005for detection of 1691 A. Procedures were exactly as previously described(Ref. 46). After hybridisation stringency washing with PR-1006 was at53° C., and with PR-1005 at 52° C.

EXAMPLE 2

We investigated the risk of venous thrombosis in individuals heterzygousand homozygous for a mutation in coagulation factor V (factor V Leiden)abnormality. We determined the factor V Leiden genotype in 471consecutive patients aged under 70 with a first objectively confirmeddeep-vein thrombosis and in 474 healthy controls. We found 85heteroqygous and 7 homozygous individuals among the cases withthrombosis, and 14 heterozygous individuals among the control subjects.

Whereas the relative risk was increased seven-fold for heterozygousindividuals, it was increased 80-fold for homozygous individuals. Theseexperienced their thrombosis at a much younger age (32 versus 44 years).The homozygous individuals were predominantly women, with mostly bloodgroup A.

Because of the increased risk of thrombosis with age, the absolute riskdifference is most pronounced in older patients, both for heterozygousand homozygous individuals. For the homozygous individuals, the absoluterisk becomes several percent per year. This implies that mostindividuals homozygous for factor V Leiden will experience at least onethrombotic even in their lifetime.

Because of the high allele frequency of the mutated factor V gene,homozygous carriers will not be extremely rare as in other types ofhereditary thrombophilia. It was unknown to data whether the homozygousstate confers a higher risk than the heterozygous state. We haveestimated the risk of thrombosis and the clinical features of patientswho were homozygous for Factor V Leiden. These were identified in alarge case-control study on deep-venous thrombosis (The LeidenThrombophilia Study: LETS) (Koster T, et al. Venous thrombosis due topoor anticoagulant response to activated protein C: Leiden ThrombophiliaStudy. Lancet 1993; 342: 1503-1506).

Methods

Study design.

The details of the design of LETS have been described previously (KosterT, et al. Venous thrombosis due to poor anticoagulant responser toactivated protein C: Leiden Thrombophilia Study. Lancet 1993; 342:1503-1506). We included consecutive patients younger than 70 years, whowere referred for the out-patient monitoring of anticoagulant treatmentto the Anticogulation Clinics of Leiden, Amsterdam and Rotterdam, aftera first, objectively confirmed episode of deep-vein thrombosis, in theabsence of known malignant disorders. Patients were seen at least sixmonths (range 6-19 months) after the acute thrombotic even. 90% ofeligible patients were willing to take part in the study. In addition to474 thrombosis patients, we included 474 control subjects who had nohistory of venous thromboembolism, did not suffer from knownmalignancies, and were of the same sex and approximately (plus/minus 5years) the same age.

Data collection and laboratory analysis.

All subjects completed a standard questionnaire, which containedquestions about the presence of acquired risk situations in the past,confined to a specific period prior to the index date, i.e. date of thethrombotic event. As acquired risk situations we considered surgery,hospitalization without surgery or prolonged immobilization at home (≧2weeks), all in the year preceding the index data, and pregnancy at thetime of the index date.

Blood was collected from the antecubital vein into Sarstedt Monovette®tubes, containing 0.106 mmol/L trisodium citrate. High-molecular-weightDNA was isolated from leucocytes and stored at 4° C. The presence of themutant factor V-Leiden gene (1691, G→A transition) was determined asdescribed. By this method we established for each patient whether he washomozygous normal (GG), heterozygous (AG) for the factor V Leidenmutation, or a homozygous carrier (AA) of this abnormality. Thetechnicians were at all times blinded to the status of the sample, i.e.whether it was from a patient or a control subject. Cells for DNAanalysis were available for 471 patients and 474 controls.

Analysis and statistics.

The frequency of heterozygous and homozygous carriers of the factor VLeiden mutation in cases and controls was compared by simplecross-tabulation. Since in the analysis of the risk associated with theheterozygous state sex and age did not appear to be confoundingvariables, as they were not expected to be for autosomal geneticabnormalities, relative risk estimates for the heterozygous state wereobtained by calculation of unmatched exposure odds ratios. A 95%confidence interval was constructed according to Woolf (Woolf B. Onestimating the relation between blood group and disease. Ann Hum Genet1955; 19:251-253).

The risk associated with the homozygous state could not be estimated inthis standard fashion, since no homozygous individuals were found amongthe controls. Therefore, under the assumption of Hardy-Weinbergequilibrium (in the controls) the expected number of homozygousindividuals in a control population was calculated, and the odds ratiowas subsequently estimated in the standard fashion. The variance of the(log) odds ratio for the homozygous state was estimated by amodification of the method of Woolf. When each cell of the two-by-twotable with cell contents a, b, c and d is considered to be therealisation of a Poisson distribution, and variance of the log(OR) is1/a+1/b+1/c+1/d (Woolf B. On estimating the relation between blood groupand disease. Ann Hum Genet 1955; 19:251-253). When the number ofindividuals with GG and AA genotypes are counted in the cases andcalculated from Hardy-Weinberg equilibrium for the controls, whichrequires a quadratic transformation, the variance of the log(OR) becomes1/AA (cases)+1/GG (cases)+4/A (controls)+4/A (control)+4/G (control), inwhich AA and GG are the number of genotypes (individuals), and A and Gthe number of alleles.

The absolute risk for thrombosis for the various genotypes and ages wascalculated by first partitioning the number of person-years in theorigin population (as derived from information from the municipalauthorities) under the assumption of Hardy-Weinberg equlibrium. Dividingthe cases in each subgroup (genotype, age) by these person-years, leadsto estimates of the absolute risks. Subsequently, these crude incidencedata were modelled after logarithmic transformation in a weighted leastsquare regression model, with three age classes (0-29: 25 yrs: 30-49: 40yrs: 50-69: 60 yrs), indicator variable for the heterozygous (0,1) andthe homozygous state (0,1), weighted for the number of cases in eachstratum. This method in which stratum-specific incidence rates are firstestimated and then smoothed ('smooth-last [Greenland S. Multivariateestimation of exposure-specific incidence from case-control studies. J.Chron. Dis. 1981; 34: 445-453]) by weighted least square regression hasbeen described by Grizzle et al (Grizzle J E, Starmer C F, Koch G G,Analysis of categorical data by linear models. Biometrics 1969; 25:489-504). Since for a Poison distribution the various of the number ofcases equals the number of cases, this is almost identical to fitting aPoisson regression model. This model will lead to more stable estimates,especially for the homozygous state, under the assumption that theincidence rate ratio for the homozygous state (and the heterozygousstate) is constant over the age strata for the log (incidence rate).This model can be written as:

Log(I)=α+β₁*age+β₂*AG(0,1)+β₃*AA(0,1), which can subsequently be used tocalculate estimates for the absolute risk and for the relative risk (asthe antilogarithm of the coefficients).

Results

Among 471 patients, we found 85% (18%) who were heterozygous and seven(1,5%) who were homozygous for the defect, whereas the other 379 (80%)did not carry the Factor V Leiden mutation. Among the 474 controls, 14(2.9%) were heterozygous and all other 460 were normal: there were nothomozygous individuals among the controls.

The homozygous individuals experienced thrombosis at a markedly youngerage than the other patients: the median age at thrombosis was 32 years,as compared to 44 years in the heterozygous, and 46 years in thepatients without the mutation (table 2).

The clinical course of the deep vein thrombosis in the homozygouspatients was unremarkable. All suffered from deep venous thrombosis ofthe leg. Four were briefly hospitalised for heparinisation, three weretreated as out-patients with cumarin derivatives only. None of the sevenpatients had a history of overt arterial disease (myocardial infarction,stroke or peripheral arterial disease) (table 3).

Six (86%) of the seven homozygous patients were women, as compared to 46(54%) of the heterozygous and 217 (57%) of the individuals without themutation. Also, six of these seven patients had blood group A, ascompared to 249 (54%) of the other 464 cases. Of the five homozygouswomen of 45 years and younger, three used oral contraceptives at thetime of the thrombotic event, which was similar to current use in allcases. Since non-O blood group and use of oral contraceptives are inthemselves risk factors for venous thrombosis, these figures indicate aninteraction between these risk factors and homozygous factor V Leidenwhich is of a multiplicative nature.

In two (29%) of the seven homozygous patients there had been apredisposing factor for thrombosis in the year preceding the event (onehad hip surgery 45 days prior to the thrombosis, and one had beenadmitted to hospital overnight after giving birth 60 days prior to thethrombotic event). Among the 85 heterozygous individuals an acquiredrisk factor had been present in 25 (29%) patients, and among the normalpatients in 131 (35%) of 379).

Previous risk situations (operations, pregnancies, hospital admissions)without thrombotic consequences were less frequent in the patientshomozygous for factor V Leiden than in the other patients. Still, fiveof the seven homozygous patients had encountered risk situations in thepast without a subsequent thrombosis (two had surgery, four had givenbirth to five children).

The seven patients were followed on average for two years withoutlong-term oral anticoagulation after the first thrombotic event. Onepatient had a recurrent thrombosis (1/13.4 yr: 7.4 percent per year). Ofthe 14 parents, three had a history of venous thrombosis, which isapproximately five times higher than expected.

Under Hardy-Weinberg equilibrium, the relative frequency ofnormals:heterozygotes:homozyzygotes is p²:2 pg:q², in which p is theallele frequency of the normal gene and q of the abnormal gene. Sincep²:2 pg was 460/474:14/474, it follows that the allele frequency offactor V Leiden (q) is 0.0.15. The allele frequencies of p=0.985 andq=0.015 conform to a distribution among 474 unselected individuals of459.9 (GG), 14.0 (AG) and 0.107 (AA).

The expected number of homozygous individuals (q²) of 0.107 among 474controls leads to an odds ration for the homozygous state of(7/379)/(0.107/460)=79. So, the risk of thrombosis for homozygousindividuals is almost eighty times increased compared to normalindividuals (CI95%: 22 to 289).

Table 4 shows the odds ratio for the three age groups, when the allelefrequency of 0.015 is used to calculate the expected number ofhomozygous controls in each age group. It is evident that the highrelative risk of thrombosis in homozygous individuals diminishes withadvancing age. This is in contrast to the relative risk for heterozygousindividuals, which is more or less constant over age.

Subsequently, we calculated absolute risks (incidence rates) for thedifferent age groups and genotypes, by using data on theage-distribution in the origin population, and under the assumption ofHardy-Weinberg equilibrium. As is shown in table 4, the incidenceincreases from only 0.55 per 10,000 per year in the youngest age groupwith GG genotype, to 16.3 per 10,000/year for heterozygous individualsin the older age groups. It is also clear from the figures in table 4,that at all age groups the risk for homozygous individuals is muchhigher than for heterozygous individuals (78 to 176 per 10,000 peryear). However, since these figures are based on only seven individualsdivided over three age groups, the estimates are unstable, and show anunexpected lower incidence in the oldest age group. The regression modelwe used smooths these estimate, since it assumes a constant relativerisk over the age groups. As FIG. 12 shows, this model fits excellentlyfor the normal and heterozygous individuals(coefficients:constant:−10.06, age: 0.0293, AG:1.96, AA:4.52). Thesmoothed incidence estimates for homozygous individuals now increasefrom 82 per 10,000 person-years in those aged under 30, to 227 per10,000 patients-years for those aged 50-69 (FIG. 13). These estimatesimply that the most homozygous patients will experience at least onethrombotic event in their lifetime.

Discussion

Resistance to APC is a common abnormality with an allele frequency forthe mutant factor V gene of about 1.5 percent. This implies that threepercent of the population is heterozygous, and homozygous individualscan be expected with a prevalence of about two per 10,000 births.

In this study we show that homozygous individuals have a high risk ofthrombosis, which is also considerably higher than the risk ofheterozygous individuals. This conclusion finds support in the young ageat which the homozygous individuals experienced their first thromboticevent.

It is clear that the risk of thrombosis in homozygous factor V Leiden isnowhere near the risk of thrombosis in homozygous protein C or protein Sdeficiency; these abnormalities lead to neonatal purpura fulminans(Branson H E, et al. Inherited protein C deficiency andcoumarin-responsive chronic relapsing purpura fulminans in a newborninfant. Lancet 1983; ii: 1165; Mahasandana C, et al. Neonatal purpurafulminans associated with homozygous protein S deficiency. Lancet 1990;335: 61-62). All of the individuals with homozygous factor V Leidenlived until adulthood before the first thrombotic event, and one evenuntil late middle age. Most of the homozygous individuals hadexperienced risk situations in the past without thrombosis, for most ofwhich (pregnancy, puerperium) no anticoagulant prophylaxis will havebeen prescribed. This shows that APC-resistance should be seen as aquantitative defect (decreased inactivation rate of factor Va) ratherthan a qualitative defect (no protein C activity) as in homozygousprotein C deficiency.

A remarkable finding in this study was the predominance of women amongthe homozygous patients. Since among these women the use of oralcontraceptives was as prevalent as among the other cases, it is likelythat this use played a role by a synergistic effect with APC-resistance.Since both pill use and APC-resistance are common, further studiesshould investigate this association, especially for the heterozygouscarriers (3 percent of all women).

The relative risk for heterozygous individuals appears constant for thedifferent age groups. This observation has to be seen in the light of abackground incidence that increases with age. This implies, as we showedin FIGS. 12 and 13 that the absolute risk of thrombosis, or the absoluterisk added by APC-resistance, becomes substantial for older heterozygousindividuals.

It may be noted that our overall estimate for the incidence rate, atabout 2 per 10,000 per year, is lower than the usual estimates of about0.5 to 1 per 1000 person-year (Branson H E, et al. Inherited protein Cdeficiency and coumarin-responsive chronic relapsing purpura fulminansin a newborn infant. Lancet 1983; ii: 1165; Koster T, More objectivediagnoses of venous thromboembolism Neth. J. Med. 1991; 38: 246-248).This is most easily explained by the age limits in our study (<70years), by the restriction to confirmed thromboses, by the exclusion ofpatients with malignancies and by the restriction to first thromboticevents.

The homozygous patients had a risk of thrombosis that was eighty timesincreased, which leads to an overall incidence of about 1 percent peryear. The observed decrease of the rate in the older age groups, may beexplained by a scarcity of individuals of that age in the population whohad not already experienced a first thrombotic event. It may also havebeen the result of the small number of homozygous patients (i.e. onlyone in the oldest age group). In both instances, the incidence figuresthat were recalculated from the weighted regression model seem the bestestimate of the risk, which becomes over two percent per year inpatients aged 50 and older.

We conclude that APC-resistance caused by homozygous factor V Leidenleads to a high risk of deep venous thrombosis. This thrombosis appearsnot to occur before aldulthood, and even does not invariably becomeapparent in risk situations such as pregnancy and puerperium. Therefore,although we are convinced that these patients should receive short-termprophylaxis with anticoagulants in risk situations. Life-longprophylaxis in individuals homozygous for factor V Leiden may howevernot necessarily be required.

TABLE 2 General characteristics of 471 thrombosis patients by factor Vgenotype GG AG AA n 379 85  7 Age median (yr)  46 44 31 range (yr-yr)15-69 17-69 22-55 Sex men (%) 162 (43) 39 (46) 1 (14) women (%) 217 (57)46 (54) 6 (86) GG: homozygous normal factor V AG: heterozygous forfactor V Leiden AA: homozygous for factor V Leiden

TABLE 3 Detailed characteristics of seven homozygous patients. Pre-Patients blood disposing factors² DVT arterial id Sex Age APC-SR groupOCC¹ 1 yr < VT intervals³ parent⁴ disease⁵  90 F 55 1.13 A — N — 0 N 124F 24 1.14 A N Y⁶ 60 0 N 266 F 30 1.20 A Y N — 0 N 173 F 22 1.14 O Y N —0 N 583 F 44 1.23 A N Y⁷ 45 1 N 589 F 42 1.21 A Y N — 1 N 944 M 31 1.19A — N — 1 N ¹use of oral contraceptives in the month preceding thethrombosis ²surgery, hospital admission, immobilisation in the yearpreceding the thrombosis, childbirth one month prior to the thrombosis,pregnancy at the time of the thrombosis ³number of days between risksituation and thrombosis ⁴number of parents with a history of venousthrombosis ⁵previous myocardial infarction, stroke or peripheralarterial disease ⁶overnight hospital stay after giving birth ⁷hipsurgery

TABLE 4 Odds ratios and absolute risk of first thrombosis by ageIncidence rates per 10⁻⁴ Cases controls Person- year¹ Age GG/AG/AA GG/AGE_(AA) OR_(AA) (CI95) OR_(AG) (CI95) year AG AG AA 0-29 61/17/2 70/3.0164 140 (1.8≡23) 6.5 (1.8≡23) 1,134,681 0.6  5.1  78.4 30-49 176/35/4217/6 .0502  98 (15≡650) 7.2 (3.0≡17) 1,006,733 1.8 11.8 176.2 50+142/33/1 173/5 .0400  30 (2.2≡429) 8.0 (3.1≡21)   682,939 2.1 16.4 110.0E_(AA) expected number of homozygous individuals among controls based onoverall Hardy-Weinberg equilibrium (q² n_(controls in age-stratum), q =.015) OR_(AA) odds ratio for homozygous individuals (versus homozygousnormals) OR_(AG) odds ratio for heterozygous individuals (versushomozygous normals) CI95 95%-confidence interval ¹crude incidence rates,based on the number of observed cases over the number of patient-years,partitioned according to Hardy-Weinberg equilibrium

FIGS. 12 and 13.

Crude (FIG. 12) and smoothed (FIG. 13) incidence rates estimates forfactor V Leiden genotypes by age.

The lowest line shows the estimates for the GG genotype, and the upperline for the AG genotype (in the figure). FIG. 13 also shows theestimates for the AA genotype (homozygous factor V Leiden). Crudeincidence estimates are indicated by +, whereas the smoothed rates areindicated by □. The smoothed incidence rates per 10,000 person-yearswere, for GG: 0.9 (0-20 yr), 1.4 (30-49 yr) and 2.5 (50-69 yr); for AG:6.3 (0-29 yr), 9.8 (30-49 yr), 17.6 (50-69 yr); for AA: 81.5 (0-29 yr),126.5 (30-49 yr) and 227.3 (50-69 yr).

EXAMPLE 3

Plasmids and in vitro RNAs

RNA isolated from PBMCs of a healthy person (homozygous wild-type) andof two patients, ID90 and ID137 (homozygous mutant, both) was obtainedfrom the Hemostatis and Thrombosis Research Centre, Leiden, theNetherlands. Fragments of 297 nt encompassing the mutation at positionamino acid 506 were cloned in the vector pG30 using the restrictionenzymes EcoRI and Csp451. The resulting plasmids were named pG30/FVwtand pG30/FVmut for wild-type and mutant clones respectively.

Cloning of the correct sequence was confirmed by sequence analysis andsubsequently the plasmids were purified by CsCl gradient for in vitroRNA synthesis. Using plasmid pG30/FVwt as a source a system controlplasmid (pG30/FV E2) was constructed by deletion of the probe sequence(21 nt) and insertion of the E2 sequence (144 nt). The 3 plasmidsdescribed above were used for in vitro RNA transcription using T7 RNApolymerase in the standard protocol. The plasmids were linearized withBamHI which, after transcription with T7 RNAP, would result in RNAsconsisting of 297 nt and 420 nt of respectively the wt, mut and systemcontrol clones followed by 700 nt of vector sequence, so that theoverall length of the in vitro RNA would be approximately 1 kb. After invitro transcription the RNA was treated with DNase I, purified using theTip 100 column (Qiagen) protocol and quantitated spectophotometrically.Appropriate serial dilutions were made in water and the in vitro RNAswere stored at −70° C.

Primers and probes.

The sequences of the NASBA amplification primers and detection probesfor ELGA and ECL detection are given in table 5.

TABLE 5 primers and probes for Factor V NASBA Length Name Sequence (nt)Remarks P1 5′ATT TCT AAT ACG ACT CAC TAT AGG 47 GAA GGT ACC AGC TTT TGTTCT CA 3′ (SEQ. ID. NO. 15) P2 5′AGT GCT TAA CAA GAC CAT ACT A 3′ 22(SEQ. ID. NO. 16) Generic 5′TGA CGT GGA CAT CAT GAG AGA 3′ 21HRP-labelled ELGA-probe (SEQ. ID. NO. 17) Generic 5′CAG CAG GCT GTG TTTGCT GTG 3′ 21 ECL-labelled ECL-probe (SEQ. ID. NO. 18) WT-probe 5′CTGGAC AGG CGA GGA ATA CAG 3′ 21 HRP-labelled (SEQ. ID. No. 19) or biotin-labelled Mut-probe 5′CTG GAC AGG CAA GGA ATA CAG 3′ 21 HRP-labelled(SEQ. ID. NO. 20) or biotin- labelled SC-probe 5′GAC ACC AAG GAA GCT TTAGAC3′ 21 biotin- (SEQ. ID. NO. 21) labelled a. The underlined part inthe P1 sequence is the T7 RNAP promoter sequence.

The P1 is located in exon 10 of the Factor V coding sequence, while theP2 sequence is located in exon 11. As a result this primer set can onlyamplify mRNA sequences from which the intron 10 sequence is removed bysplicing. Due to better performance in either ELGA or ECL there are twogeneric probes. However, it should be possible to choose one genericprobe for both ELGA and ECL. The amplification primers were purified on20% acryl amide, 7M urea slab gels. After elution and EtOH precipitationthe primers were dissolved in 500 μl H₂O and the concentrationdetermined by spectophotometry (OD260).

Biotin oligos were made on the synthesizer and used after EtOHprecipitation and dissolving in H₂O. Coupling the HRP label toNH₂-oligos was done according to the standard protocol and the probe wasused without further purification (generic ELGA probe) or purified on aslab gel (wild-type and mutant specific ELGA probes). The ECL oligoswere synthesized and used without further purification.

Nucleic acid isolation.

All nucleic acid isolations were performed using the method described byBoom et al. (1990, J. Clin. Microbiol 28: 495-503). Nucleic acid wasextracted from 100 μl whole blood (see clinical samples) and elution wasin 100 μl H₂O, typically 5 μl of eluate was used as input for NASBAamplification. The remainder of the eluate was stored at −70° C.

NASBA amplifications.

NASBA amplifications were performed as follows. To 5 μl of RNA 18 μl ofa premix solution was added that consisted of: 10 μl 2.5×NRG buffer(final concentration in 1×buffer: 40 mM tris, pH=8.5, 70 mM KCl, 1 mMeach dNTP, 2 mM ATP/CTP/UTP, 1.5 mM GTP, 0.5 mM ITP, 12 mM MgCl₂), 6.25μl 4×primer mix (final concentration in 1×buffer: 15% v/v DMSO, 0.2 μMP1 and 0.2 μM P2) and 1.75 μl H₂O. The sample was incubated at 65° C.for 5 minutes and subsequently incubated at 41° C. for 5 minutes.Leaving the tubes as much as possible at 41° C. 2 μl enzyme mix (8 unitsAMV-RT, 40 units T7 RNAP, 0.1 unit E. coli RNase H, 2.6 μg BSA, 1.5 Msorbitol) was added, followed by gentle mixing (i.e. tapping) andincubation at 41° C. for 90 minutes.

ELGA detection.

For ELGA detection 3 different probe solutions were used containing ageneric probe, wild-type probe and mutant probe respectively. In orderto increase the specificity of the wild-type and mutant HRP labelledprobes, these labelled probes were mixed with their counterpartnon-labelled probe (see table 6).

TABLE 6 Ratios of labelled and non-labelled probes to increase thespecificity of hybridization for ELGA detection Amplificate toHRP-labelled probe non-labelled probe detect (molec/hyb) (molec/hyb)Generic^(a)) ELGA Generic (2 × 10¹⁰) — Wild-type Wild-type (2 × 11)^(b))Mutant (5 × 10¹³) Mutant Mutant (2 × 10¹¹)^(b)) Wild-type (2 × 10¹³)^(a))This includes: wild-type, mutant and system control amplificates^(b))Due to the purification process (slab gel) the specific activity ofthe wild-type and mutant HRP probes is lower than normal.

After amplification 1 μl of amplificate was added to 4 μl of appropriateprobe mix (final concentration in 5 μl: 1×SSC, BFB, XCFF, 5% v/vglycerol and the appropriate probes, see table 2), mixed and incubatedfor 15 minutes at 45° C. Subsequently 2.5 μl of the sample was analyzedon an acrylamide gel (5% acryl/bisacryl, 0.04% dextrane sulphate, NASBAelfo buffer=25 mM tris, 25 mM boric acid, 500 μM EDTA, pH=8.3) run at150 V in 0.5×NASBA elfo buffer. After electrophoresis the gel wasstained using the standard TMB/UP substrate solutions (mixed at 1:1ratio) for approximately 6 minutes. Usually the gels were fixed in 50%methanol (0/N) and air dried between 2 sheets of transparent foil.

ECL detection.

For ECL also 3 different probe solutions were used for detection ofamplificate (see table 7).

TABLE 7 Ratios of labelled and non-labelled probes to increase thespecificity of hybridization for ECL detection. Non-labelled AmplificateECL-labelled Biotin capture probe to detect probe (molec/hyb) probe(molec/hyb) (molec/hyb) Wild-type ECL generic Wild-type Mutant (2 ×10¹²) (2 × 10¹²)^(a)) (2 × 10¹³) Mutant ECL generic Mutant Wild-type (2× 10¹²) (2 × 10¹²)^(a)) (8 × 10¹²) System control ECL generic SC — (2 ×10¹²) (2 × 10¹²) ^(a))Streptavidin coated beads for 100 hybridisation(200 μl Dynal beads) reactions were loaded with 2 × 10³⁴ moleculesbiotin capture probes.

To set up the hybridisation reaction 10 μl ECL mix (0.1% w/v BSA,12.5×SSC, 2×10¹² molecules ECL generic probe), 10 μl bead mix (0.1 w/vBSA, 1×PBS, 2 μl appropriate bead solution and the appropriatenon-labelled probe) and 5 μl 21 fold diluted (in water) amplificate weremixed and incubated for 30 minutes at 45° C. under constant shaking in astove. Subsequently 300 μl ECL assay buffer was added and the tubesplaced in the ECL instrument for reading of ECL signals.

Results

Sensitivity.

The primers used for NASBA amplification of the Factor V mRNA generatean amplificate of 182 nt long for the wild-type and mutant sequence.When the primers are used for amplification of the system control (SC)in vitro RNA the result is an amplimer of 305 nt in length. Thesensitivity of the amplification was investigated using serial dilutionsof in vitro generated wild-type, mutant and SC RNA (Table 8).

TABLE 8 Sensitivity of the Factor V mRNA NASBA using ELGA detection withthe generic ELGA probe Amount ELGA Input RNA (molecules) resultsWild-type 10⁴ + 10³ + 10² + 10¹ − 10⁰ − Mutant 10⁴ + 10³ + 10² + 10¹ +10⁰ − SC 10⁴ + 10³ + 10² + 10¹ ± 10⁰ −

For all 3 input RNAs the analytical sensitivity is at least 100molecules. The reactions with an input of 10 molecules are occasionallypositive, indicating that the sensitivity is actually between 10 and 100molecules.

EXAMPLE 4

The methods used are as described for example 3.

In order to determine the amount of SC RNA that should be spiked in thenucleic acid isolation without competing with the wild-type of mutantRNA when present, several amounts of SC RNA were analyzed. These SC RNAamounts were isolated without addition of sample, with the addition of100 μl whole blood and as a control a dilution series of SC RNA wasdirectly amplified. The results of ELGA analysis after amplification aredepicted in table 5. Apparently there is some loss of nucleic acidduring nucleic acid isolation (compare lanes 3 with and withoutisolation, A and C, respectively).

From the A series (table 9) it can be concluded that the minimum amountof SC RNA that should be spiked in the lysisbuffer is 1×10⁵ molecules.This amount of SC RNA is not inhibitory for the amplification ofwild-type or mutant RNA isolated from 100 μl whole blood. In fact, evenwhen 10 times more SC RNA is used, this is not inhibitory for wild-typeor mutant RNA isolated from 100 μl whole blood (table 5, B series). Inall further experiments, when appropriate, 10⁵ molecules of SC RNA werespiked in the lysisbuffer before nucleic acid isolation.

TABLE 9 ELGA results of amplification of different amount of Systemcontrol RNA spiked before nucleic acid isolation Wt/Mut Input Set-up SCsignal signal 1 A + − 2 A + − 3 A − − 4 A − − 1 B − + 2 B − + 3 B − + 4B − + 1 C + − 2 C + − 3 C + − 4 C − − ELGA results of amplification ofdifferent amount of CS RNA spiked before nucleic acid isolation ANucleic acid isolation without addition of sample B Nucleic acidisolation with 100 μl whole blood C control, direct amplification of SCRNA 1 1 × 10⁶ molecules SC RNA in lysis buffer (± 5 × 10⁴ peramplification) 2 1 × 10⁴ molecules SC RNA in lysisbuffer (± 5 × 10³ peramplification) 3 1 × 10⁴ molecules SC RNA in lysisbuffer (± 5 × 10² peramplification) The probe used for hybridization was the generic ELGAprobe.

EXAMPLE 5

The methods used are as described in example 3

Due to the nature of the mutation in the Factor V mRNA, a G→A singlebase mutation, it is expected that the wild-type probe will giveconsiderable background signal on mutant amplificate and vice versa.This will be the case for both ELGA and ECL detection. In order to avoidcomplicated hybridization protocols the labelled probes are mixed withtheir non-labelled counterpart to suppress background hybridization onthe non-homologous amplificate. In table 10 the results of specificdetection of wild-type and mutant amplificates with probe mixtures aredepicted, using ELGA detection.

TABLE 10 ELGA detection of wild-type mutant amplificates with specificwild-type (WT) and mutant (Mut) probes labelled with HRP. LabelledNon-labelled Excess non- probe probe labelled probe WT-signal Mut signalWT Mut 1 ++ ++ 5 ++ ++ 10 ++ + 100 ++ ± 250 ++ − 500 + − Mut WT 1 + ++5 + ++ 10 ± ++ 100 − ++ 250 − ± WT = wild-type HRP-labelled probe; Mut =mutant HRP-labelled probe

It is apparent that in case of the wild-type HRP labelled probe a 250fold excess of non-labelled mutant probe should be added to reduce thebackground sufficiently. When the HRP-labelled mutant probe is used, anexcess of 100 fold non-labelled wild-type probe is sufficient to reducethe background to an acceptable level. A more or less identicalexperiment was performed using ECL detection. In the ECL method thenon-labelled probe has to compete with the specific biotinylated captureprobe on the magnetic bead. The result of the ECL detection usingdifferent excess non-labelled probe ratios is depicted in Table 11.

TABLE 11 ECL detection of wild-type and mutant amplificates withspecific wild-type (WT) and mutant (Mut) probes. Non- Excess non-Labelled labelled labelled WT-signal Mut signal probe probe probe(x1000) (x1000) WT Mut 0 620 250 1 250 20 2.5 300 1 5 200 1 10 100 0 MutWT 0 300 600 1 2 200 2.5 3 100 5 0 80 10 0 30 WT = wild-typeHRP-labelled probe; Mut = mutant HRP-labelled probe

For subsequent ECL detection an excess of 10 fold non-labelled mutantprobe with biotinylated wild-type probe on beads and an excess of 4 foldnon-labelled wild-type probe with biotinylated mutant probe on beads wasused. The differences between amount of non-labelled probe that has tobe added for ELGA and ECL detection has to do with the hybridizationformats. In the ECL format the specific probe is bound to the magneticbead and therefore will have slower hybridization kinetics compared toprobes in solution. As a result a relatively small excess of in solutionnon-labelled probe has to be added. In the ELGA the competition takesplace between 2 probes in solution, which makes it necessary to add arelatively high amount of non-labelled probe.

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14 6909 base pairs nucleic acid both unknown cDNA 1 GAATTCCGCAGCCCGGAGTG TGGTTAGCAG CTCGGCAAGC GCTGCCCAGG TCCTGGGGTG 60 GTGGCAGCCAGCGGGAGCAG GAAAGGAAGC ATGTTCCCAG GCTGCCCACG CCTCTGGGTC 120 CTGGTGGTCTTGGGCACCAG CTGGGTAGGC TGGGGGAGCC AAGGGACAGA AGCGGCACAG 180 CTAAGGCAGTTCTACGTGGC TGCTCAGGGC ATCAGTTGGA GCTACCGACC TGAGCCCACA 240 AACTCAAGTTTGAATCTTTC TGTAACTTCC TTTAAGAAAA TTGTCTACAG AGAGTATGAA 300 CCATATTTTAAGAAAGAAAA ACCACAATCT ACCATTTCAG GACTTCTTGG GCCTACTTTA 360 TATGCTGAAGTCGGAGACAT CATAAAAGTT CACTTTAAAA ATAAGGCAGA TAAGCCCTTG 420 AGCATCCATCCTCAAGGAAT TAGGTACAGT AAATTATCAG AAGGTGCTTC TTACCTTGAC 480 CACACATTCCCTGCGGAGAA GATGGACGAC GCTGTGGCTC CAGGCCGAGA ATACACCTAT 540 GAATGGAGTATCAGTGAGGA CAGTGGACCC ACCCATGATG ACCCTCCATG CCTCACACAC 600 ATCTATTACTCCCATGAAAA TCTGATCGAG GATTTCAACT CGGGGCTGAT TGGGCCCCTG 660 CTTATCTGTAAAAAAGGGAC CCTAACTGAG GGTGGGACAC AGAAGACGTT TGACAAGCAA 720 ATCGTGCTACTATTTGCTGT GTTTGATGAA AGCAAGAGCT GGAGCCAGTC ATCATCCCTA 780 ATGTACACAGTCAATGGATA TGTGAATGGG ACAATGCCAG ATATAACAGT TTGTGCCCAT 840 GACCACATCAGCTGGCATCT GCTGGGAATG AGCTCGGGGC CAGAATTATT CTCCATTCAT 900 TTCAACGGCCAGGTCCTGGA GCAGAACCAT CATAAGGTCT CAGCCATCAC CCTTGTCAGT 960 GCTACATCCACTACCGCAAA TATGACTGTG GGCCCAGAGG GAAAGTGGAT CATATCTTCT 1020 CTCACCCCAAAACATTTGCA AGCTGGGATG CAGGCTTACA TTGACATTAA AAACTGCCCA 1080 AAGAAAACCAGGAATCTTAA GAAAATAACT CGTGAGCAGA GGCGGCACAT GAAGAGGTGG 1140 GAATACTTCATTGCTGCAGA GGAAGTCATT TGGGACTATG CACCTGTAAT ACCAGCGAAT 1200 ATGGACAAAAAATACAGGTC TCAGCATTTG GATAATTTCT CAAACCAAAT TGGAAAACAT 1260 TATAAGAAAGTTATGTACAC ACAGTACGAA GATGAGTCCT TCACCAAACA TACAGTGAAT 1320 CCCAATATGAAAGAAGATGG GATTTTGGGT CCTATTATCA GAGCCCAGGT CAGAGACACA 1380 CTCAAAATCGTGTTCAAAAA TATGGCCAGC CGCCCCTATA GCATTTACCC TCATGGAGTG 1440 ACCTTCTCGCCTTATGAAGA TGAAGTCAAC TCTTCTTTCA CCTCAGGCAG GAACAACACC 1500 ATGATCAGAGCAGTTCAACC AGGGGAAACC TATACTTATA AGTGGAACAT CTTAGAGTTT 1560 GATGAACCCACAGAAAATGA TGCCCAGTGC TTAACAAGAC CATACTACAG TGACGTGGAC 1620 ATCATGAGAGACATCGCCTC TGGGCTAATA GGACTACTTC TAATCTGTAA GAGCAGATCC 1680 CTGGACAGGCGAGGAATACA GAGGGCAGCA GACATCGAAC AGCAGGCTGT GTTTGCTGTG 1740 TTTGATGAGAACAAAAGCTG GTACCTTGAG GACAACATCA ACAAGTTTTG TGAAAATCCT 1800 GATGAGGTGAAACGTGATGA CCCCAAGTTT TATGAATCAA ACATCATGAG CACTATCAAT 1860 GGCTATGTGCCTGAGAGCAT AACTACTCTT GGATTCTGCT TTGATGACAC TGTCCAGTGG 1920 CACTTCTGTAGTGTGGGGAC CCAGAATGAA ATTTTGACCA TCCACTTCAC TGGGCACTCA 1980 TTCATCTATGGAAAGAGGCA TGAGGACACC TTGACCCTCT TCCCCATGCG TGGAGAATCT 2040 GTGACGGTCACAATGGATAA TGTTGGAACT TGGATGTTAA CTTCCATGAA TTCTAGTCCA 2100 AGAAGCAAAAAGCTGAGGCT GAAATTCAGG GATGTTAAAT GTATCCCAGA TGATGATGAA 2160 GACTCATATGAGATTTTTGA ACCTCCAGAA TCTACAGTCA TGGCTACACG GAAAATGCAT 2220 GATCGTTTAGAACCTGAAGA TGAAGAGAGT GATGCTGACT ATGATTACCA GAACAGACTG 2280 GCTGCAGCATTAGGAATTAG GTCATTCCGA AACTCATCAT TGAACCAGGA AGAAGAAGAG 2340 TTCAATCTTACTGCCCTAGC TCTGGAGAAT GGCACTGAAT TCGTTTCTTC GAACACAGAT 2400 ATAATTGTTGGTTCAAATTA TTCTTCCCCA AGTAATATTA GTAAGTTCAC TGTCAATAAC 2460 CTTGCAGAACCTCAGAAAGC CCCTTCTCAC CAACAAGCCA CCACAGCTGG TTCCCCACTG 2520 AGACACCTCATTGGCAAGAA CTCAGTTCTC AATTCTTCCA CAGCAGAGCA TTCCAGCCCA 2580 TATTCTGAAGACCCTATAGA GGATCCTCTA CAGCCAGATG TCACAGGGAT ACGTCTACTT 2640 TCACTTGGTGCTGGAGAATT CAGAAGTCAA GAACATGCTA AGCGTAAGGG ACCCAAGGTA 2700 GAAAGAGATCAAGCAGCAAA GCACAGGTTC TCCTGGATGA AATTACTAGC ACATAAAGTT 2760 GGGAGACACCTAAGCCAAGA CACTGGTTCT CCTTCCGGAA TGAGGCCCTG GGAGGACCTT 2820 CCTAGCCAAGACACTGGTTC TCCTTCCAGA ATGAGGCCCT GGGAGGACCC TCCTAGTGAT 2880 CTGTTACTCTTAAAACAAAG TAACTCATCT AAGATTTTGG TTGGGAGATG GCATTTGGCT 2940 TCTGAGAAAGGTAGCTATGA AATAATCCAA GATACTGATG AAGACACAGC TGTTAACAAT 3000 TGGCTGATCAGCCCCCAGAA TGCCTCACGT GCTTGGGGAG AAAGCACCCC TCTTGCCAAC 3060 AAGCCTGGAAAGCAGAGTGG CCACCCAAAG TTTCCTAGAG TTAGACATAA ATCTCTACAA 3120 GTAAGACAGGATGGAGGAAA GAGTAGACTG AAGAAAAGCC AGTTTCTCAT TAAGACACGA 3180 AAAAAGAAAAAAGAGAAGCA CACACACCAT GCTCCTTTAT CTCCGAGGAC CTTTCACCCT 3240 CTAAGAAGTGAAGCCTACAA CACATTTTCA GAAAGAAGAC TTAAGCATTC GTTGGTGCTT 3300 CATAAATCCAATGAAACATC TCTTCCCACA GACCTCAATC AGACATTGCC CTCTATGGAT 3360 TTTGGCTGGATAGCCTCACT TCCTGACCAT AATCAGAATT CCTCAAATGA CACTGGTCAG 3420 GCAAGCTGTCCTCCAGGTCT TTATCAGACA GTGCCCCCAG AGGAACACTA TCAAACATTC 3480 CCCATTCAAGACCCTGATCA AATGCACTCT ACTTCAGACC CCAGTCACAG ATCCTCTTCT 3540 CCAGAGCTCAGTGAAATGCT TGAGTATGAC CGAAGTCACA AGTCCTTCCC CACAGATATA 3600 AGTCAAATGTCCCCTTCCTC AGAACATGAA GTCTGGCAGA CAGTCATCTC TCCAGACCTC 3660 AGCCAGGTGACCCTCTCTCC AGAACTCAGC CAGACAAACC TCTCTCCAGA CCTCAGCCAC 3720 ACGACTCTCTCTCCAGAACT CATTCAGAGA AACCTTTCCC CAGCCCTCGG TCAGATGCCC 3780 ATTTCTCCAGACCTCAGCCA TACAACCCTT TCTCCAGACC TCAGCCATAC AACCCTTTCT 3840 TTAGACCTCAGCCAGACAAA CCTCTCTCCA GAACTCAGTC AGACAAACCT TTCCCCAGCC 3900 CTCGGTCAGATGCCCCTTTC TCCAGACCTC AGCCATACAA CCCTTTCTCT AGACTTCAGC 3960 CAGACAAACCTCTCTCCAGA ACTCAGCCAT ATGACTCTCT CTCCAGAACT CAGTCAGACA 4020 AACCTTTCCCCAGCCCTTGG TCAGATGCCC ATTTCTCCAG ACCTCAGCCA TACAACCCTT 4080 TCTCTAGACTTCAGCCAGAC AAACCTCTCT CCAGAACTCA GTCAAACAAA CCTTTCCCCA 4140 GCCCTCGGTCAGATGCCCCT TTCTCCAGAC CCCAGCCATA CAACCCTTTC TCTAGACCTC 4200 AGCCAGACAAACCTCTCTCC AGAACTCAGT CAGACAAACC TTTCCCCAGA CCTCAGTGAG 4260 ATGCCCCTCTTTGCAGATCT CAGTCAAATT CCCCTTACCC CAGACCTCGA CCAGATGACA 4320 CTTTCTCCAGACCTTGGTGA GACAGATCTT TCCCCAAACT TTGGTCAGAT GTCCCTTTCC 4380 CCAGACCTCAGCCAGGTGAC TCTCTCTCCA GACATCAGTG ACACCACCCT TCTCCCGGAT 4440 CTCAGCCAGATATCACCTCC TCCAGACCTT GATCAGATAT TCTACCCTTC TGAATCTAGT 4500 CAGTCATTGCTTCTTCAAGA ATTTAATGAG TCTTTTCCTT ATCCAGACCT TGGTCAGATG 4560 CCATCTCCTTCATCTCCTAC TCTCAATGAT ACTTTTCTAT CAAAGGAATT TAATCCACTG 4620 GTTATAGTGGGCCTCAGTAA AGATGGTACA GATTACATTG AGATCATTCC AAAGGAAGAG 4680 GTCCAGAGCAGTGAAGATGA CTATGCTGAA ATTGATTATG TGCCCTATGA TGACCCCTAC 4740 AAAACTGATGTTAGGACAAA CATCAACTCC TCCAGAGATC CTGACAACAT TGCAGCATGG 4800 TACCTCCGCAGCAACAATGG AAACAGAAGA AATTATTACA TTGCTGCTGA AGAAATATCC 4860 TGGGATTATTCAGAATTTGT ACAAAGGGAA ACAGATATTG AAGACTCTGA TGATATTCCA 4920 GAAGATACCACATATAAGAA AGTAGTTTTT CGAAAGTACC TCGACAGCAC TTTTACCAAA 4980 CGTGATCCTCGAGGGGAGTA TGAAGAGCAT CTCGGAATTC TTGGTCCTAT TATCAGAGCT 5040 GAAGTGGATGATGTTATCCA AGTTCGTTTT AAAAATTTAG CATCCAGACC GTATTCTCTA 5100 CATGCCCATGGACTTTCCTA TGAAAAATCA TCAGAGGGAA AGACTTATGA AGATGACTCT 5160 CCTGAATGGTTTAAGGAAGA TAATGCTGTT CAGCCAAATA GCAGTTATAC CTACGTATGG 5220 CATGCCACTGAGCGATCAGG GCCAGAAAGT CCTGGCTCTG CCTGTCGGGC TTGGGCCTAC 5280 TACTCAGCTGTGAACCCAGA AAAAGATATT CACTCAGGCT TGATAGGTCC CCTCCTAATC 5340 TGCCAAAAAGGAATACTACA TAAGGACAGC AACATGCCTG TGGACATGAG AGAATTTGTC 5400 TTACTATTTATGACCTTTGA TGAAAAGAAG AGCTGGTACT ATGAAAAGAA GTCCCGAAGT 5460 TCTTGGAGACTCACATCCTC AGAAATGAAA AAATCCCATG AGTTTCACGC CATTAATGGG 5520 ATGATCTACAGCTTGCCTGG CCTGAAAATG TATGAGCAAG AGTGGGTGAG GTTACACCTG 5580 CTGAACATAGGCGGCTCCCA AGACATTCAC GTGGTTCACT TTCACGGCCA GACCTTGCTG 5640 GAAAATGGCAATAAACAGCA CCAGTTAGGG GTCTGGCCCC TTCTGCCTGG TTCATTTAAA 5700 ACTCTTGAAATGAAGGCATC AAAACCTGGC TGGTGGCTCC TAAACACAGA GGTTGGAGAA 5760 AACCAGAGAGCAGGGATGCA AACGCCATTT CTTATCATGG ACAGAGACTG TAGGATGCCA 5820 ATGGGACTAAGCACTGGTAT CATATCTGAT TCACAGATCA AGGCTTCAGA GTTTCTGGGT 5880 TACTGGGAGCCCAGATTAGC AAGATTAAAC AATGGTGGAT CTTATAATGC TTGGAGTGTA 5940 GAAAAACTTGCAGCAGAATT TGCCTCTAAA CCTTGGATCC AGGTGGACAT GCAAAAGGAA 6000 GTCATAATCACAGGGATCCA GACCCAAGGT GCCAAACACT ACCTGAAGTC CTGCTATACC 6060 ACAGAGTTCTATGTAGCTTA CAGTTCCAAC CAGATCAACT GGCAGATCTT CAAAGGGAAC 6120 AGCACAAGGAATGTGATGTA TTTTAATGGC AATTCAGATG CCTCTACAAT AAAAGAGAAT 6180 CAGTTTGACCCACCTATTGT GGCTAGATAT ATTAGGATCT CTCCAACTCG AGCCTATAAC 6240 AGACCTACCCTTCGATTGGA ACTGCAAGGT TGTGAGGTAA ATGGATGTTC CACACCCCTG 6300 GGTATGGAAAATGGAAAGAT AGAAAACAAG CAAATCACAG CTTCTTCGTT TAAGAAATCT 6360 TGGTGGGGAGATTACTGGGA ACCCTTCCGT GCCCGTCTGA ATGCCCAGGG ACGTGTGAAT 6420 GCCTGGCAAGCCAAGGCAAA CAACAATAAG CAGTGGCTAG AAATTGATCT ACTCAAGATC 6480 AAGAAGATAACGGCAATTAT AACACAGGGC TGCAAGTCTC TGTCCTCTGA AATGTATGTA 6540 AAGAGCTATACCATCCACTA CAGTGAGCAG GGAGTGGAAT GGAAACCATA CAGGCTGAAA 6600 TCCTCCATGGTGGACAAGAT TTTTGAAGGA AATACTAATA CCAAAGGACA TGTGAAGAAC 6660 TTTTTCAACCCCCCAATCAT TTCCAGGTTT ATCCGTGTCA TTCCTAAAAC ATGGAATCAA 6720 AGTATTACACTTCGCCTGGA ACTCTTTGGC TGTGATATTT ACTAGAATTG AACATTCAAA 6780 AACCCCTGGAAGAGACTCTT TAAGACCTCA AACCATTTAG AATGGGCAAT GTATTTTACG 6840 CTGTGTTAAATGTTAACAGT TTTCCACTAT TTCTCTTTCT TTTCTATTAG TGAATAAAAT 6900 TTTATACAA6909 20 base pairs nucleic acid both unknown cDNA 2 TGCTGACTATGATTACCAGA 20 20 base pairs nucleic acid both unknown cDNA 3 GAGTAACAGATCACTAGGAG 20 20 base pairs nucleic acid both unknown cDNA 4 GCATTTACCCTCATGGAGTG 20 25 base pairs nucleic acid both unknown cDNA 5 CAAGAGTAGTTATGCTCTCA GGCAC 25 20 base pairs nucleic acid both unknown cDNA 6CACGTGGTTC ACTTTCACGG 20 24 base pairs nucleic acid both unknown cDNA 7TGTGGTATAG CAGGACTTCA GGTA 24 18 base pairs nucleic acid both unknowncDNA 8 TATAAGATCC ACCATTGT 18 22 base pairs nucleic acid both unknowncDNA 9 TGCCCAGTGC TTAACAAGAC CA 22 20 base pairs nucleic acid bothunknown cDNA 10 TGTTATCACA CTGGTGCTAA 20 22 base pairs nucleic acid bothunknown cDNA 11 GAGAGACATC GCCTCTGGGC TA 22 18 base pairs nucleic acidboth unknown cDNA 12 TGGACAGGCG AGGAATAC 18 18 base pairs nucleic acidboth unknown cDNA 13 TGGACAGGCA AGGAATAC 18 149 base pairs nucleic acidboth unknown cDNA 14 GTATTTTGTC CTTGAAGTAA CCTTTCAGAA ATTCTGAGAATTTCTTCTGG CTAGAACATG 60 TTAGGTCTCC TGGCTAAATA ATGGGGCATT TCCTTCAAGAGAACAGTAAT TGTCAAGTAG 120 TCCTTTTTAG CACCAGTGTG ATAACATTT 149

What is claimed is:
 1. A method for determining whether a test person ishomozygotic or heterozygotic for a mutation in Factor V and/or Factor Vaor Factor VIII and/or Factor VIIIa comprising carrying out a method fordetermining whether a defect is present in response to APC, comprisingadding APTT reagent and CaCl₂ in an amount of more than 25 mM to asample to be tested and further carrying out determination of a valuefor a parameter known to be useful for diagnosis of the defect, andcomparing the value obtained with a value obtained in the same mannerfor a sample from a normal individual or from an individual known to behomozygotic or heterozygotic, thereby establishing whether the testperson is homozygotic or heterozygotic for a mutation in Factor V and/orFactor Va or Factor VIII and/or Factor VIIIa.
 2. A method fordetermining whether a test person is homozygotic or heterozygotic for amutation in Factor V and/or Factor Va or Factor VIII and/or Factor VIIIacomprising carrying out a method for determining whether a defect ispresent in anticoagulant response to APC, comprising adding APTT reagentand CaCl₂ in an amount of more than 25 mM to a sample to be tested andfurther carrying out determination of a value for a parameter known tobe useful for diagnosis of the defect, and ascertaining the test personis abnormal when the result is a value below 0.84, to thereby establishwhether the test person is homozygotic or heterozygotic for a mutationin Factor V and/or Factor Va or Factor VIII and/or Factor VIIIa.
 3. Amethod of diagnosis of an increased risk of a thrombotic eventcomprising carrying out a method according to claims 1 or
 2. 4. Themethod according to claim 1, wherein said mutation is in Factor V and/orFactor Va.
 5. The method according to claim 1, wherein said mutation isin Factor V.
 6. The method according to claim 1, wherein said mutationis in Factor Va.
 7. The method according to claim 1, wherein saidmutation is in Factor VIII and/or Factor VIIIa.
 8. The method accordingto claim 1, wherein said mutation is in Factor VIII.
 9. The methodaccording to claim 1, wherein said mutation is in Factor VIIIa.
 10. Themethod according to claim 2, wherein said mutation is in Factor V and/orFactor Va.
 11. The method according to claim 2, wherein said mutation isin Factor V.
 12. The method according to claim 2, wherein said mutationis in Factor Va.
 13. The method according to claim 2, wherein saidmutation is in Factor VIII and/or Factor VIIIa.
 14. The method accordingto claim 2, wherein said mutation is in Factor VIII.
 15. The methodaccording to claim 2, wherein said mutation is in Factor VIIIa.